Inhibitory effect and mechanism of oregano essential oil on Listeria monocytogenes cells, toxins and biofilms.

Listeria monocytogenes (L. monocytogenes) is a prevalent foodborne pathogen with a remarkable capacity to form biofilms on utensil surfaces. The Listeriolysin O (LLO) exhibits hemolytic activity, which is responsible for causing human infections. In this study, we investigated the inhibitory effect and mechanism of oregano essential oil (OEO) on L. monocytogenes, evaluated the effects on its biofilm removal and hemolytic activity. The minimum inhibitory concentration (MIC) of OEO against L. monocytogenes was 0.03% (v/v). L. monocytogenes was treated with OEO at 3/2 MIC for 30 min the bacteria was decreased below the detection limit (10 CFU/mL) in PBS and TSB (the initial bacterial load was about 6.5 log CFU/mL). The level of L. monocytogenes in minced pork co-cultured with OEO (15 MIC) about 2.5 log CFU/g lower than that in the untreated group. The inhibitory mechanisms of OEO against planktonic L. monocytogenes encompassed perturbation of cellular morphology, elevation in reactive oxygen species levels, augmentation of lipid oxidation extent, hyperpolarization of membrane potential, and reduction in intracellular ATP concentration. In addition, OEO reduced biofilm coverage on the surface of glass slides by 62.03% compared with the untreated group. Meanwhile, OEO (1/8 MIC) treatment reduced the hemolytic activity of L. monocytogenes to 24.6% compared with the positive control. Molecular docking suggested carvacrol and thymol might reduce the hemolytic activity of L. monocytogenes. The results of this study demonstrate that OEO exhibits inhibitory effects against L. monocytogenes, biofilms and LLO, which had potential as natural antimicrobial for the inhibition of L. monocytogenes.

Prevalence, serotype, antimicrobial susceptibility, contamination factors, and control methods of Salmonella spp. in retail fresh fruits and vegetables: A systematic review and meta-analysis.

This research presents a comprehensive review of Salmonella presence in retail fresh fruits and vegetables from 2010 to 2023, utilizing data from recognized sources such as PubMed, Scopus, and Web of Science. The study incorporates a meta-analysis of prevalence, serovar distribution, antimicrobial susceptibility, and antimicrobial resistance genes (ARGs). Additionally, it scrutinizes the heterogeneous sources across various food categories and geographical regions The findings show a pooled prevalence of 2.90% (95% CI: 0.0180-0.0430), with an increase from 4.63% in 2010 to 5.32% in 2022. Dominant serovars include S. Typhimurium (29.14%, 95% CI: 0.0202-0.6571) and S. Enteritidis (21.06%, 95% CI: 0.0181-0.4872). High resistance rates were noted for antimicrobials like erythromycin (60.70%, 95% CI: 0.0000-1.0000) and amoxicillin (39.92%, 95% CI: 0.0589-0.8020). The most prevalent ARGs were blaTEM (80.23%, 95% CI: 0.5736-0.9692) and parC mutation (66.67%, 95% CI: 0.3213-0.9429). Factors such as pH, water activity, and nutrient content, along with external factors like the quality of irrigation water and prevailing climatic conditions, have significant implications on Salmonella contamination. Nonthermal sterilization technologies, encompassing chlorine dioxide, ozone, and ultraviolet light, are emphasized as efficacious measures to control Salmonella. This review stresses the imperative need to bolster prevention strategies and control measures against Salmonella in retail fresh fruits and vegetables to alleviate related food safety risks.

Prevalence, antibiotic susceptibility, and genomic analysis of Vibrio alginolyticus isolated from seafood and freshwater products in China.

Introduction: This study characterized Vibrio alginolyticus isolated from seafood and freshwater products in China (2020). Methods and Results: In total, 122 (95.31%) V. alginolyticus isolates were resistant to at least 1 antibiotic category, and 2 (1.56%) isolates were resistant to at least 3 antibiotic categories and belong to multi-drug resistance (MDR) isolates. A high prevalence rate was observed to be blaCARB (98.04%) encoding beta-lactam resistance, followed by tet (97.06%) encoding tetracycline resistance and fos (4.90%) encoding resistance to fosfomycin. Among the 57 V. alginolyticus isolates, the commonest virulence genes were type III secretion system translocated gene vopD, vopB, and vcrH (54.4%, 31/57), type III secretion system regulated gene tyeA (54.39%), followed by vscI and vscF (50.88%) encoded type III secretion system inner rod protein and needle protein, respectively. Multilocus sequence typing (MLST) showed considerable genetic diversity, with 34 distinct sequence types (STs) identified among 55 isolates. ST421 (n = 5), ST166 (n = 4), ST523 (n = 3), ST516 (n = 3), and ST507 (n = 3) were dominant STs among 55 V. alginolyticus isolates. Discussion: These findings highlight the widespread occurrence of V. alginolyticus in both freshwater and seafood products, underscoring the critical need for vigilant monitoring of these bacteria. Such measures are essential for ensuring effective food safety management and safeguarding public health.

The role of DsbA and PepP genes in the environmental tolerance and virulence factors of Cronobacter sakazakii.

Cronobacter sakazakii, an opportunity foodborne pathogen, could contaminate a broad range of food materials and cause life-threatening symptoms in infants. The bacterial envelope structure contribute to bacterial environment tolerance, biofilm formation and virulence in various in Gram-negative bacteria. DsbA and PepP are two important genes related to the biogenesis and stability of bacterial envelope. In this study, the DsbA and PepP were deleted in C. sakazakii to evaluate their contribution to stress tolerance and virulence of the pathogen. The bacterial environment resistance assays showed DsbA and PepP are essential in controlling C. sakazakii resistance to heat and desiccation in different mediums, as well as acid, osmotic, oxidation and bile salt stresses. DsbA and PepP also played an important role in regulating biofilm formation and motility. Furthermore, DsbA and PepP deletion weaken C. sakazakii adhesion and invasion in Caco-2, intracellular survival and replication in RAW 264.7. qRT-PCR results showed that DsbA and PepP of C. sakazakii played roles in regulating the expression of several genes associated with environment stress tolerance, biofilm formation, bacterial motility and cellular invasion. These findings indicate that DsbA and PepP played an important regulatory role in the environment resisitance, biofilm formation and virulence of C. sakazakii, which enrich understanding of genetic determinants of adaptability and virulence of the pathogen.

Prevalence and characterization of foodborne pathogens isolated from fresh-cut fruits and vegetables in Beijing, China.

Pre-cut fresh fruits and vegetables are highly appealing to consumers for their convenience, however, as they are highly susceptible to microbial contamination in processing, the potential risks of foodborne illnesses to public health are not negligible. This study aimed to assess the prevalence, antibiotic susceptibility and molecular characteristics of major foodborne pathogens (Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella) isolated from fresh-cut fruits and vegetables in Beijing, China. 86 stains were isolated from 326 samples, with S. aureus being the highest prevalence (15.38 %), followed by E. coli (9.23 %) and L. monocytogenes (1.85 %), while no Salmonella was detected. The prevalence by type of food indicated that fruit trays and mixed vegetables were more susceptible to contamination by pathogens. 98 % of S. aureus were resistant to at least of one antibiotic, and showed a high resistance rate to benzylpenicillin (90 %) and oxacillin (48 %). Among 25 E. coli isolates, 57.67 % of which exhibited multi-drug resistance, with common resist to trimethoprim/sulfamethoxazole (66.67 %) and ampicillin (63.33 %). A total of 9 sequence types (STs) and 8 spa types were identified in 35 S. aureus isolates, with ST398-t34 being the predominant type (42.86 %). Additionally, analysis of 25 E. coli isolates demonstrated significant heterogeneity, characterized by 22 serotypes and 18 STs. Genomic analysis revealed that 5 and 44 distinct antibiotic resistance genes (ARGs) in S. aureus and E. coli, respectively. Seven quinolone resistance-determining regions (QRDRs) mutations were identified in E. coli isolates, of which GyrA (S83L) was the most frequently detected. All the S. aureus and E. coli isolates harbored virulence genes. ARGs in S. aureus and E. coli showed a significant positive correlation with plasmids. Furthermore, one L. monocytogenes isolate, which was ST101 and serogroupIIc from watermelon sample, harbored virulence genes (inlA and inlB) and LIPI-1 pathogenic islands (prfA, plcA, hly and actA), which posed potential risks for consumer's health. This study focused on the potential microbial risk of fresh-cut fruits and vegetables associated with foodborne diseases, improving the scientific understanding towards risk assessment related to ready-to-eat foods.

Antimicrobial susceptibility and genomic characterization of Vibrio parahaemolyticus isolated from aquatic foods in 15 provinces, China, 2020.

Prevalent in marine, estuarine and coastal environments, Vibrio parahaemolyticus is one of the major foodborne pathogens which can cause acute gastroenteritis through consumption of contaminated food. This study encompassed antimicrobial resistance, molecular characteristics and phylogenetic relationships of 163 V. parahaemolyticus isolated from aquatic foods across 15 provinces in China. The isolates showed high resistance rates against ampicillin (90.80 %, 148/163) and cefazolin (72.39 %, 118/163). Only 5 isolates demonstrated multi-drug resistance (MDR) phenotypes. A total of 37 different antibiotic resistance genes (ARGs) in correlation with seven antimicrobial categories were identified. tet(34) and tet(35) were present in all 163 isolates. Other most prevalent ARGs were those conferring resistance to ß-lactams, with prevalence rate around 18.40 % (30/163). The virulence genes tdh and trh were found in 17 (10.43 %) and 9 (5.52 %) isolates, respectively. Totally 121 sequence types (STs) were identified through whole genome analysis, among which 60 were novel. The most prevalent sequence type was ST3 (9.20 %, 15/163), which shared the same genotype profile of trh_, tdh+ and blaCARB-22+. Most of the tdh+V. parahaemolyticus isolates was clustered into a distinctive clade by the phylogenetic analysis. Our study showed that the antimicrobial resistance of V. parahaemolyticus in aquatic foods in China was moderate. However, the emerging of MDR isolates implicate strengthened monitoring is needed for the better treatment of human V. parahaemolyticus infections. High genetic diversity and virulence potential of the isolates analyzed in this study help better understanding and evaluating the risk of V. parahaemolyticus posed to public health.

Total coliforms, microbial diversity and multiple characteristics of Salmonella in soil-irrigation water-fresh vegetable system in Shaanxi, China.

Global occurrences of foodborne disease outbreaks have been documented, involving fresh agricultural produce contaminated by various pathogens. This contamination can occur at any point in the supply chain. However, studies on the prevalence of total coliforms, Salmonella and microbial diversity in vegetable and associated environments are limited. This study aimed to assess 1) the number of total coliforms (n = 299) and diversity of microbial communities (n = 52); 2) the prevalence, antibiotic susceptibility, genomic characteristics, and potential transmission relationships of Salmonella in soil-irrigation water-vegetable system (n = 506). Overall, 84.28 % samples were positive to total coliforms, with most frequently detected in soil (100 %), followed by irrigation water (79.26 %) and vegetables (62.00 %). A seasonal trend in coliform prevalence was observed, with significantly higher levels in summer (P < 0.05). Detection rates of Salmonella in soil, vegetable and irrigation water were 2.21 %, 4.74 % and 9.40 %. Fourteen serotypes and sequence types (STs) were respectively annotated in 56 Salmonella isolates, ST13 S. Agona (30.36 %, 17/56), ST469 S. Rissen (25.00 %, 14/56), and ST36 S. Typhimurium (12.50 %, 7/56) were dominant serotypes and STs. Thirty-one (55.36 %) isolates were multi-drug resistant, and the resistance was most frequently found to ampicillin (55.36 %, 31/56), followed by to sulfamethoxazole (51.79 %, 29/56) and tetracycline (50.00 %, 28/56). The genomic characteristics and antibiotic resistance patterns of Salmonella isolates from soil, vegetables, and irrigation water within a coherent geographical locale exhibited remarkable similarities, indicating Salmonella may be transmitted among these environments or have a common source of contamination. Microbial alpha diversity indices in soil were significantly higher (P < 0.05) than that in vegetable and irrigation water. The microbial phylum in irrigation water covered that in the vegetable, demonstrating a significant overlap in the microbial communities between the vegetables and the irrigation water.

Pre-Harvest Food Safety Challenges in Food-Animal Production in Low- and Middle-Income Countries.

Food safety remains a significant global public health concern, with the risk of unsafe food varying worldwide. The economies of several low- and middle-income countries (LMICs) heavily rely on livestock, posing a challenge to ensuring the production of safe food. This review discusses our understanding of pre-harvest critical issues related to food safety in LMICs, specifically focusing on animal-derived food. In LMICs, food safety regulations are weak and inadequately enforced, primarily concentrating on the formal market despite a substantial portion of the food sector being dominated by informal markets. Key critical issues at the farm level include animal health, a low level of good agriculture practices, and the misuse of antimicrobials. Effectively addressing foodborne diseases requires a comprehensive One Health framework. Unfortunately, the application of the One Health approach to tackle food safety issues is notably limited in LMICs. In conclusion, considering that most animal-source foods from LMICs are marketed through informal channels, food safety legislation and policies need to account for this context. Interventions aimed at reducing foodborne bacterial pathogens at the farm level should be scalable, and there should be strong advocacy for the proper implementation of pre-harvest interventions through a One Health approach.

Revealing the mechanism of citral induced entry of Vibrio vulnificus into viable but not culturable (VBNC) state based on transcriptomics.

Citral has attracted much attention as a safe and effective plant-derived bacteriostatic agent. However, the ability of citral to induce the formation of VBNC state in Vibrio vulnificus has not been evaluated. In the present study, V. vulnificus was shown to be induced to form the VBNC state at 4.5 h and 3 h of citral treatment at 4MIC and 6MIC. Moreover, the citral-induced VBNC state of V. vulnificus maintained some respiratory chain activity and was able to recover well in both APW media, APW media supplemented with 5 % (v/v) Tween 80 and 2 mg/mL sodium pyruvate. Field emission and transmission electron microscopy showed that the external structure of the citral-induced VBNC V. vulnificus cells was shortened to short rods, with folded cell membrane, rough cell surface, and dense cytoplasm and loose nuclear material in the internal cell structure. In addition, the possible molecular mechanisms of citral-induced formation and recovery of V. vulnificus in the VBNC state were explored by transcriptomics. Transcriptome analyses revealed that 1118 genes were significantly altered upon entry into the VBNC state, and 1052 genes were changed after resuscitation. Most of the physiological activities related to energy production were inhibited in the citral-induced VBNC state of V. vulnificus; however, the bacteria retained its pathogenicity. The citral-induced resuscitation of V. vulnificus in the VBNC state selectively restored the activity of some genes related to bacterial growth and reproduction. Meanwhile, the expression levels of other genes may have been influenced by citral-induced resuscitation after the formation of the VBNC state. In conclusion, this study evaluated and analyzed the ability and possible mechanism of citral on the formation of VBNC state and the recovery of VBNC state of V. vulnificus, and made a comprehensive assessment for the safety of citral application in food production.

Antibiotic susceptibility and genomic analysis of ciprofloxacin-resistant and ESBLs-producing Escherichia coli in vegetables and their irrigation water and growing soil.

The rise of antibiotic resistance in Escherichia coli has become a major global public health concern. While there is extensive research on antibiotic-resistant E. coli from human and animal sources, studies on vegetables and their environments are limited. This study investigated the prevalence and characteristics of ciprofloxacin-resistant (CIPR) E. coli in 13 types of edible raw vegetables, along with their irrigation water and soil in Shaanxi, China. Of 349 samples collected (157 vegetables, 59 water, and 133 soil), a total of 48 positive samples were detected, with one CIPRE. coli strain isolated from each sample being selected for further analyses. A striking observation was its high prevalence in irrigation water at 44.1 %, markedly exceeding that in vegetables (12.0 %) and soil (4.5 %). The susceptibility of Forty-eight CIPRE. coli isolates was evaluated using the disc diffusion method for 18 different antibiotics, all these isolates were not only resistant to the tested fluoroquinolones antibiotics (levofloxacin, nalidixic acid), but also displayed a multi-drug resistance (MDR) pattern. Twenty-eight (58.3 %) of 48 CIPRE. coli isolates exhibited extended spectrum ß-lactamases (ESBLs) (CIPR-ESBLs) producing phenotype. Subsequently, whole-genome sequencing was performed on these 28 isolates. We identified 12 serotypes and STs each, with O101: H9 (35.7 %, 10/28) and ST10 (21.4 %, 6/28) being the most common. Further classification placed these isolates into five phylogenetic groups: A (57.1 %, 16/28), B1 (32.1 %, 9/28), D (3.6 %, 1/28), B2 (3.6 %,1/28), and F (3.6 %,1/28). Notelly, Identical ST types, serotypes and phylogroups were found in certain CIPR-ESBLs-producing E. coli from both vegetables and adjacent irrigation water. Genomic analysis of the 28 CIPR-ESBLs-producing E. coli isolates unveiled 73 resistance genes, associated with 13 amino acid mutations in resistance-determining regions (QRDRs) and resistance to 12 types of antibiotics. Each isolate was confirmed to carry both ESBLs and fluoroquinolone resistance genes, with the Ser83Ala mutation in GyrA (96.4 %, 27/28) being the most prevalent. A detailed analysis of Mobile Genetic Elements (MGEs) revealed that IncFIB and IncFII plasmid subtypes were most prevalent in 60.7 % and 67.9 % of isolates, respectively, with 75 % containing over 10 insertion sequences (IS) each. Furthermore, we observed that certain ESBL and PMQR genes were located on plasmids or in proximity to insertion sequences. In conclusion, our research highlights the widespread presence of CIPRE. coli in irrigation water and thoroughly examines the genetic characteristics of CIPR-ESBLs-producing E. coli strains, underlining the need for ongoing monitoring and management to reduce multidrug-resistant bacteria in vegetables and their environment.

Synergistic bactericidal effect and mechanism of ultrasound combined with Lauroyl Arginate Ethyl against Salmonella Typhimurium and its application in the preservation of onions.

In the present study, the synergistic bactericidal effect and mechanism of ultrasound (US) combined with Lauroyl Arginate Ethyl (LAE) against Salmonella Typhimurium were investigated. On this basis, the effect of US+LAE treatment on the washing of S. Typhimurium on the surface of onions and on the physical and chemical properties of onion during fresh-cutting and storage were studied. The results showed that treatment with US+LAE could significantly (P < 0.05) reduce the number of S. Typhimurium compared to US and LAE treatments alone, especially the treatment of US+LAE (230 W/cm2, 8 min, 71 µM) reduced S. Typhimurium by 8.82 log CFU/mL. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein and nucleic acid release and N-phenyl-l-naphthylamine (NPN) assays demonstrated that US+LAE disrupted the integrity and permeability of S. Typhimurium cell membranes. Reactive oxygen species (ROS) and malondialdehyde (MDA) assays indicated that US+LAE exacerbated oxidative stress and lipid peroxidation in cell membranes. Field emission scanning electron microscopy (FESEM) demonstrated that US+LAE treatment caused loss of cellular contents and led to cell crumpling and even lost the original cell morphology. US+LAE treatment caused a significant (P < 0.05) decrease in the number of S. Typhimurium on onions, but there was no significant (P > 0.05) effect on the color, hardness, weight and ascorbic acid content of onions. This study elucidated the synergistic antibacterial mechanism of US+LAE and verified the feasibility of bactericidal effect on the surface of onions, providing a theoretical basis for improving the safety of fresh produce in the food industry and to propose a new way to achieve the desired results.

Extraction and characterization of polysaccharide from fermented mycelia of Coriolus versicolor and its efficacy for treating nonalcoholic fatty liver disease.

Coriolus versicolor, a popular traditional Chinese medicinal herb, is widely used in China to treat spleen and liver diseases; however, the beneficial effects of C. versicolor polysaccharides (CVPs) on nonalcoholic fatty liver disease (NAFLD) remain elusive. Herein we isolated and purified a novel CVP (molecular weight, 17,478 Da) from fermented mycelium powder. This CVP was composed of mannose, galacturonic acid, glucose, galactose, xylose, and fucose at a molar ratio of 22:1:8:15:10:3. Methylation, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses indicated that the CVP backbone consisted of →1)-ß-D-Man-(6,4→1)-α-D-Gal-(3→1)-α-D-Man-(4→1)-α-D-Gal-(6→, with branches of →1)-α-D-Glc-(6→1)-α-D-Man-(4,3→1)-ß-D-Xyl-(2→1)-ß-D-Glc on the O-6 position of →1)-ß-D-Man-(6,4→ of the main chain. The secondary branches linked to the O-4 position of →1)-α-D-Man-(4,3→ with the chain of →1)-α-D-Fuc-(4→1)-α-D-Man. Further, CVP treatment alleviated the symptoms of NAFLD in an HFD-induced mice model. CVP altered gut microbiota, predominantly suppressing microbes associated with bile acids both in the serum and cecal contents. In vitro data showed that CVP reduced HFD-induced hyperlipidemia via farnesoid X receptor. Our results improve our understanding of the mechanisms underlying the cholesterol- and lipid-lowering effects of CVP and indicate that CVP is a promising candidate for NAFLD therapy.

Prevalence, antibiotic susceptibility and genomic analysis of Salmonella from retail meats in Shaanxi, China.

Salmonella is a major foodborne pathogen that poses a substantial risk to food safety and public health. This study aimed to assess the prevalence, antibiotic susceptibility, and genomic features of Salmonella isolates recovered from 600 retail meat samples (300 pork, 150 chicken and 150 beef) from August 2018 to October 2019 in Shaanxi, China. Overall, 40 (6.67 %) of 600 samples were positive to Salmonella, with the highest prevalence in chicken (21.33 %, 32/150), followed in pork (2.67 %, 8/300), while no Salmonella was detected in beef. A total of 10 serotypes and 11 sequence types (STs) were detected in 40 Salmonella isolates, with the most common being ST198 S. Kentucky (n = 15), ST13 S. Agona (n = 6), and ST17 S. Indiana (n = 5). Resistance was most commonly found to tetracycline (82.50 %), followed by to ampicillin (77.50 %), nalidixic acid (70.00 %), kanamycin (57.50 %), ceftriaxone (55.00 %), cefotaxime (52.50 %), cefoperazone (52.50 %), chloramphenicol (50.00 %), levofloxacin (57.50 %), cefotaxime (52.50 %), kanamycin (52.50 %), chloramphenicol (50.00 %), ciprofloxacin (50.00 %), and levofloxacin (50.00 %). All ST198 S. Kentucky isolates showed multi-drug resistance (MDR; ≥3 antimicrobial categories) pattern. Genomic analysis showed 56 distinct antibiotic resistance genes (ARGs) and 6 target gene mutations of quinolone resistance determining regions (QRDRs) in 40 Salmonella isolates, among which, the most prevalent ARG types were related to aminoglycosides and ß-lactams resistance, and the most frequent mutation in QRDRs was GyrA (S83F) (47.5 %). The number of ARGs in Salmonella isolates showed a significant positive correlation with the numbers of insert sequences (ISs) and plasmid replicons. Taken together, our findings indicated retail chickens were seriously contaminated, while pork and beef are rarely contaminated by Salmonella. Antibiotic resistance determinants and genetic relationships of the isolates provide crucial data for food safety and public health safeguarding.

Microbiological analysis and characterization of Salmonella and ciprofloxacin-resistant Escherichia coli isolates recovered from retail fresh vegetables in Shaanxi Province, China.

Fresh vegetables are closely associated with foodborne disease outbreaks; however, systematic analysis of the microbiological quality of fresh vegetables and molecular information on foodborne pathogens in fresh produce are poorly reported in China. Here, we evaluated the epidemiological prevalence of coliforms via the most probable number method and characterized Salmonella and ciprofloxacin-resistant (CIPR) Escherichia coli isolates recovered from retail fresh vegetables in Shaanxi Province, China. Antimicrobial susceptibility testing, serotype determination, multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST), antibiotic resistance encoding gene (ARG) annotation, virulence factor prediction, and functional classification were performed. Between October 2020 and September 2021, 576 samples (i.e., tomatoes, lettuces, spinaches, and cabbages) were found to be positive for coliforms, and the prevalence of coliforms showed a seasonal trend. Coliform counts of vegetables in supermarkets in Xi'an were significantly lower (P < 0.01) than that in other cities. The detection rates of Salmonella and CIPRE. coli-positive vegetables were 1 % (6/576) and 0.7 % (4/576), respectively. All isolates exhibited resistance to ≥1 antibiotics, and 92.9 % (13/14) were multidrug-resistant. One extended spectrum ß-lactamase (ESBL)-producing CIPRE. coli isolate in spinach was resistant to not only three third-generation cephalosporins but also to two polymyxins. Among nine Salmonella isolates, five different serovars (S. Enteritidis, S. Indiana, monophasic variant of S. Typhimurium, S. Agona, and S. Gallinarum), four sequence types (STs; ST11, ST13, ST17, and ST34), and seven core genome STs (cgSTs) were identified. Five CIPRE. coli strains were assigned to three serovars (O101:H4, O8:H18, and O11:H25), three STs (ST44, ST48, and ST457), and four cgSTs. Coexisting amino acid mutations of Thr57Ser/Ser80Arg in ParC and Ser83Phe/Asp87Gly in GyrA in quinolone resistance-determining regions (QRDRs) might be causes for nalidixic acid resistance. Eight definite virulence profiles in eight serovars were identified. Notably, cdtB and pltA only encoded typhoid toxins and were just detected from S. Typhoid isolates were also detected from S. Indiana and monophasic S. Typhimurium, which are closely associated with swine food chain were first detected in fresh vegetables. In conclusion, our findings suggest that coliform contamination on fresh vegetables is prevalent in this province. Most Salmonella and CIPRE. coli isolates were phenotypically and genetically diverse and could resist multiple antibiotics by carrying multiple ARGs and virulence genes.

Effects of Wheat Tempering with Slightly Acidic Electrolyzed Water on the Microbiota and Flour Characteristics.

Slightly acidic electrolyzed water (SAEW) was prepared and used as wheat tempering water. This study explored the impacts of tempering with SAEW on microbial load and diversity and quality properties of wheat flour. As SAEW volume ratio increased, the residual level of total plate counts (TPC) and mould/yeast counts (MYC) decreased dramatically (p < 0.05). Based on genomics analysis, bacterial 16S rRNA gene and fungal ITS1 gene region were performed to characterize the changes in microbial communities' composition and diversity in response to SAEW treatment. SAEW optimal volume ratio (6.5:10, v/v) of SAEW with distilled water influenced wheat microbiome composition, with a higher microbial diversity and abundance discovered on the control grains. Bacteroidetes of predominant bacterial phylum and Ascomycota of the most abundant fungal phylum were reduced after SAEW optimal volume ratio tempering. The flour yield is higher and ash content is lower than the control samples. Falling number and "b*" in terms of colour markedly increased. DSC (Differential Scanning Calorimetry) test showed that To (onset temperature), Tp (peak temperature), and Tc (conclusion temperature) were significantly decreased in thermal characteristics of flour. Gluten content, protein content, ΔH and pasting properties tests showed no significant change. It can be concluded that SAEW should be applied on wheat tempering for producing clean wheat flour. ANOVA and Tukey's honestly significant difference (HSD) test were used for the analysis of variance and differences between the experimental and control groups, with p < 0.05.

Effects of Broussonetia papyrifera (L.) L'Hér. ex Vent. fruits water extract on hippocampal neurogenesis in the treatment of APP/PS1 transgenic mice.

Background: Adult neurogenesis plays an important role in repairing damaged neurons and improving cognitive impairment in Alzheimer's disease (AD). B. Papyrifera (L.) L'Hér. ex Vent. fruits (BL), a traditional Chinese medicine for tonifying the kidney, has been reported to improve cognitive function in AD mice, but the underlying mechanisms have not been clearly illuminated. This study aimed to provide an overview of the differential compounds in the brain of APP/PS1 mice after BL water extract (BLWE) treatment through metabolomics technology and to elucidate whether the therapeutic effect and mechanism are through the enhancement of neurogenesis. Methods: APP/PS1 transgenic mice were treated with different doses of BLWE. After 6 weeks of intragastric injection, the therapeutic effects of BLWE on APP/PS1 transgenic mice were determined by the Morris water maze test, immunohistochemistry, hematoxylin & eosin and Nissl staining, enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Subsequently, metabolomics technology was used to analyze the regulatory effect of BLWE on differential compounds in the brain of APP/PS1 mice, and on this basis, its molecular mechanism of BLWE was screened. Finally, the protein expression of the Wnt/ß-catenin signaling pathway was detected by Western blotting. Results: After BLWE treatment, the learning and memory function of APP/PS1 mice were significantly improved, which was related to the increase in the number of Nestin+/BrdU+ and NeuN+/BrdU+ cells, and the decrease in the number of apoptotic cells in the hippocampus. BLWE treatment could also up-regulate the expression of synapse-associated proteins. Moreover, BLWE could modulate endogenous metabolic compounds in the brains of AD mice, including N-acetyl-aspartate, glutamine, etc. Furthermore, BLWE inhibited the phosphorylation of Tyr216-GSK-3ß and ß-catenin protein while increased CyclinD1 protein expression. Conclusion: We demonstrated that BLWE can enhance neural stem cells proliferation and improve neurogenesis, thereby efficiently repairing damaged neurons in the hippocampus and ameliorating cognitive impairment in APP/PS1 transgenic mice. The mechanism is at least partly through activating the Wnt/ß-catenin signaling pathway.

ESBLs-producing Escherichia coli from sheep-origin: Genome-wide virulence genes identification and in vivo virulence assessment in mice and Galleria mellonella.

The worldwide spread of pathogenic Escherichia coli, together with the multidrug resistant linked with extended-spectrum ß-lactamases (blaCTX-M , blaTEM and blaOXA ), not only affect the health of animals and humans but also bring huge economic losses to animal husbandry. Despite the high levels of virulence present in many extended-spectrum beta-lactamases (ESBLs)-producing E. coli isolates, however, few studies have comprehensively assessed the pathogenicity of ESBLs-producing E. coli isolates. Thus, the aim of the present study was to investigate the presence of virulence genes in third-generation cephalosporin-resistant E. coli and to assess their pathogenicity and zoonotic potential. Previously, we identified 67 ESBLs-producing E. coli strains from sheep anal swabs in northwest China. In this study, we genotypically and phenotypically characterized isolates of E. coli that produce ESBLs. According to the VirulenceFinder and virulence factors database, all ESBLs-producing E. coli strains harboured a wide range of virulence genes. The ColV plasmid-related genes (hlyF, ompT, iss, iutA and cvaC) were present in 52 (77.6%) ESBLs-producing E. coli isolates. Surprisingly, quite a number of extraintestinal pathogenic E. coli virulence-related genes were detected in 62 (92.5%) of 67 isolates. A total of 33 serotypes and 37 sequence types (STs) were found in 67 ESBLs-producing isolates. ST10 is the most prevalent ST, which is represented by five strains. The cluster analysis showed that CC10 and CC23 were the common clonal complexes (CCs). Predominant serotypes were O8 (10%) and O9 (9%) followed by 6% each of O89, O101 and O185. Most sheep-origin ESBLs-producing E. coli held the highly pathogenic to human and displayed moderate-to-vigorous-intensity motor capacity. The ESBLs-producing E. coli isolates with numerous virulence-related genes were able to cause multiple infectious diseases in animal models (mice, neonatal rats and Galleria mellonella). To our knowledge, this study represents an important first step for a comprehensive characterization of pathogenicity and zoonotic potential of sheep-origin ESBLs-producing E. coli isolates. These findings may be of significant value for the identification of pathogenicity and zoonotic potential risks associated with sheep-origin ESBLs-producing E. coli.

Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates That Cause Diarrhea in Sheep in Northwest China.

Development of extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli is one the greatest threats faced by mankind. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants (i.e., sheep and goats) in China. The aim of this study was to identify and characterize the resistance profiles, resistomes, and sequence features of 67 ESBL-producing E. coli isolates from sheep in northwest China. The findings showed that blaCTX-M and blaTEM were the most prevalent. Interestingly, we found that the resistance gene mcr-1 was widespread in sheep merely from Shaanxi areas, accounting for 19.2% (5/26). The highly prevalent serotypes and FumC-FimH (CH) typing isolates were O8 and C4H32, respectively. High-risk E. coli clones, such as sequence type 10 (ST10), ST23, ST44, and ST58, were also found in China's sheep population. A total of 67 ESBL-producing isolates were divided into five phylogenetic groups, namely, B1 (n = 47, 70.1%), B2 (n = 1, 1.5%), C (n = 14, 20.9%), E (n = 1, 1.5%), and F (n = 1, 1.5%), with the phylogenetic groups for 3 isolates (4.5%) remaining unknown. Moreover, ESBL-producing E. coli isolates were also characterized by the abundance and diversity of biocide/metal resistance genes and insert sequences. We found that in ESBL-producing E. coli isolates, there were two different types of isolates, those containing ESBL genes or not, which led to large discrepancies between resistance phenotypes and resistomes. In summary, our study provides a comprehensive overview of resistance profiles and genome sequence features in ESBL-producing E. coli and highlights the possible role of sheep as antibiotic resistance gene disseminators into humans. IMPORTANCE Antimicrobial resistance (AMR), especially the simultaneous resistance to several antibiotics (multidrug resistance [MDR]), is one of the greatest threats to global public health in the 21st century. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants in China. This study is the largest and most comprehensive analysis of ESBL-producing E. coli isolates from sheep, including antibiotic resistance profiles, phylogenetic groups, serotypes, multilocus sequence types (MLST), insert sequences (IS), antibiotic resistance genes, disinfectant resistance genes, and heavy metal resistance genes. We recommend extending the surveillance of AMR of sheep-origin E. coli to prevent future public health risks.

Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut.

Since mcr-1 was first discovered in 2015, this gene has shown excellent transmission ability and evolutionary characteristics worldwide, leading to major public health and food safety concerns. In this study, chicken meat was used as a food vehicle for the conjugation of mcr-1-bearing Salmonella at different storage temperatures (4 °C, 25 °C, and 37 °C) to simulate mcr-1 transmission during food transportation and storage and determine its efficiency and mechanism. In addition, conjugation experiments were performed in mouse gut to further confirm that mcr-1 is horizontally transferred in vivo during food consumption. 16S rDNA sequencing of mouse stool samples was performed to understand the effect of horizontal transfer of mcr-1 on mouse gut bacteria. mcr-1-bearing plasmids were characterized using pulsed-field gel electrophoresis (PFGE) and S1 nuclease-PFGE and sequenced by Illumina sequencing. Our results showed that mcr-1-bearing plasmids in donors are successfully transferred to recipients on chicken meat at not only 25 °C and 37 °C but also 4 °C with conjugation frequencies between 1.32 × 10-6 and 3.85 × 10-4 per recipient cell. In mouse gut, mcr-1 was transferred not only to the recipient bacteria introduced by intragastric administration but also to the intestinal bacteria (E. coli strain named as E6353). Horizontal transfer of mcr-1-bearing plasmid in mouse gut negatively affected the mouse intestinal microbiota. In a constant conjugative environment, plasmid replicon type is the most decisive factor affecting the conjugation frequency. The peak number of transconjugants in group D6-E. coli C600 with an IncHI2-type mcr-1-bearing plasmid (1.43 × 102 colony-forming units [CFU]/g feces) was significantly higher than that of transconjugants in group D7-E. coli C600 with an IncX4-type mcr-1-bearing plasmid (0.3 × 102 CFU/g feces). The upstream and downstream genetic environment of mcr-1 in different plasmid replicon types in Salmonella varied during conjugation in different horizontal transfer environments. An IncI2 plasmid (p25-D4R7S1_mcr-1) lost the insertion sequence ISApl1, which originally existed upstream of mcr-1, when this plasmid transferred from donor to recipient cells on chicken meat at 25 °C. An IncHI2 plasmid was more active than IncI2 and IncX4 plasmids during bacterial reproduction and evolution; an IncFIB-IncHI2 hybrid plasmid (p6176253_mcr-1) was formed in mouse gut during conjugation from pD6_tet(M) and pD6_mcr-1. mcr-1 is captured by mobile genetic elements IS26 in IncX4 plasmids and ISApl1 in IncI2, IncHI2, and IncFIB-IncHI2 hybrid plasmids and is disseminated among bacteria.

Effects of 405 ± 5-nm LED Illumination on Environmental Stress Tolerance of Salmonella Typhimurium in Sliced Beef.

Salmonella Typhimurium is a widely distributed foodborne pathogen and is tolerant of various environmental conditions. It can cause intestinal fever, gastroenteritis and bacteremia. The aim of this research was to explore the effect of illumination with 405 nm light-emitting diodes (LEDs) on the resistance of S. Typhimurium to environmental stress. Beef slices contaminated with S. Typhimurium were illuminated by 405 nm LEDs (18.9 ± 1.4 mW/cm2) for 8 h at 4 °C; controls were incubated in darkness at 7 °C. Then, the illuminated or non-illuminated (control) cells were exposed to thermal stress (50, 55, 60 or 65 °C); oxidative stress (0.01% H2O2 [v/v]); acid stress (simulated gastric fluid [SGF] at pH 2 or 3); or bile salts (1%, 2%, or 3% [w/v]). S. Typhimurium treated by 405 nm LED irradiation showed decreased resistance to thermal stress, osmotic pressure, oxidation, SGF and bile salts. The transcription of eight environmental tolerance-related genes were downregulated by the illumination. Our findings suggest the potential of applying 405 nm LED-illumination technology in the control of pathogens in food processing, production and storage, and in decreasing infection and disease related to S. Typhimurium.