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In this study, the impact of different discharge time, discharge power, and storage time on the pH, oxidation-reduction potential (ORP), electrical conductivity (EC), H2O2, and the physicochemical properties and the stability at 4°C were evaluated. With the increase of plasma discharge time and discharge power, all the values increased except pH value. There were no significant changes in the values of pH, ORP, and EC for 30 days storage at 4°C (p > 0.05). The concentrations of NO3−, NO2−, and H2O2 exhibited significant changes (p < 0.05) after stored for 5 days. The plasma discharge time and discharge power showed significant effects on the physicochemical properties and activity of PAW. Moreover, the concentrations of NO3− and NO2− significantly affected the storage stability of PAW at 4°C.

ABSTRACT: Objectives: The study aimed to characterize the quinolone resistance of Salmonella enterica serovar Typhimurium and its monophasic variant (Salmonella enterica serovar 1,4,[5],12:i:-) isolated from food and patients in China. Methods: All of the isolates were assessed for quinolone susceptibility via the broth microdilution method. Then, the isolates were checked for mutations within quinolone resistance-determining regions of gyrA, gyrB, parC, and parE and were examined for plasmid-mediated quinolone resistance genes. Results: High rates of resistance to nalidixic acid in the S. Typhimurium (70.7%) and S. 1,4,[5],12:i:- (41.9%) isolates were observed, and a considerable proportion of isolates with reduced susceptibility to ciprofloxacin and levofloxacin were also detected. The high frequency of mutations in GyrA (60.8%) and a variety of genes (aac[6′]-Ib-cr [23.2%], oqxAB [19.2%], qnrS [13.6%], and qnrA [3.2%]) conferring quinolone resistance in these Salmonella isolates were noteworthy. Lastly, the isolates carrying qnrS for transferability and transmission of the quinolone resistance were analysed by conjugation. Multiple locus variable-number tandem repeat analysis profiles indicated that some qnrS-positive isolates were clonally related, whilst the other isolates were genetically divergent. This suggested that both clonal spread of resistant strains and horizontal transmission of the plasmid-mediated resistance genes contributed to the dissemination of qnrS-positive Salmonella isolates. Conclusion: This study highlights the prevalence of quinolone-resistant S. Typhimurium and S. 1,4,[5],12:i:- in China, posing a threat to public health.

Background: The issue of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLR) has consistently posed challenges for clinical treatment. Hence, a rapid susceptibility testing (AST) method urgently needs to be developed. Methods: In the present study, 35 isolates of H. pylori were isolated from 203 gastritis patients of the Guangzhou cohort, and the antimicrobial resistance phenotypes were associated with their genomes to analyze the relevant mutations. Based on these mutations, a rapid detection system utilizing high-resolution melting (HRM) curve analysis was designed and verified by the Shenzhen cohort, which consisted of 38 H. pylori strains. Results: Genomic analysis identified the mutation of the 2143 allele from A to G (A2143G) of 23S rRNA as the most relevant mutation with CLR resistance (p < 0.01). In the HRM system, the wild-type H. pylori showed a melting temperature (Tm) of 79.28 ± 0.01 °C, while the mutant type exhibited a Tm of 79.96 ± 0.01 °C. These differences enabled a rapid distinction between two types of H. pylori (p < 0.01). Verification examinations showed that this system could detect target DNA as low as 0.005 ng/μL in samples without being affected by other gastric microorganisms. The method also showed a good performance in the Shenzhen validation cohort, with 81.58% accuracy, and 100% specificity. Conclusions: We have developed an HRM system that can accurately and quickly detect CLR resistance in H. pylori. This method can be directly used for the detection of gastric microbiota samples and provides a new benchmark for the simple detection of H. pylori resistance.

Abstract Background Changes in the gut microbiota composition is a hallmark of chronic kidney disease (CKD), and interventions targeting the gut microbiota present a potent approach for CKD treatment. This study aimed to evaluate the efficacy and safety of washed microbiota transplantation (WMT), a modified faecal microbiota transplantation method, on the renal activity of patients with renal dysfunction. Methods A comparative analysis of gut microbiota profiles was conducted in patients with renal dysfunction and healthy controls. Furthermore, the efficacy of WMT on renal parameters in patients with renal dysfunction was evaluated, and the changes in gut microbiota and urinary metabolites after WMT treatment were analysed. Results Principal coordinate analysis revealed a significant difference in microbial community structure between patients with renal dysfunction and healthy controls (P = 0.01). Patients with renal dysfunction who underwent WMT exhibited significant improvement in serum creatinine, estimated glomerular filtration rate, and blood urea nitrogen (all P

This study aimed to compare the nutritive value and obesity prevention of ordinary Cordyceps militaris (CM) and selenium-enriched CM (SeCM). The results indicated that Se enrichment significantly increased the total carbohydrate and soluble dietary fiber content, while the protein and insoluble dietary fiber content decreased. Although the fat content was not affected, the medium and long-chain fatty acids content significantly changed. Moreover, Se enrichment significantly elevated the secondary metabolites belonging to terpenoids and alkaloids, which are linked with the enhanced biosynthesis of secondary metabolites. Both CM and SeCM reduced body weight, adipose accumulation, impaired glucose tolerance, and lipid levels in high-fat diet (HFD)-fed mice, and there was no significant difference between them. Network pharmacological analysis revealed that dietary CM and SeCM prevented HFD-induced obesity and associated metabolic diseases with multi-ingredients acting on multi-targets. Overall, Se enrichment improved the nutritive value of CM without altering its role in preventing obesity.

Rapid, sensitive, point-of-care detection of pathogenic bacteria is important for food safety. In this study, we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip (QBs-labelled LFIAS) combined with strand displacement loop-mediated isothermal amplification (SD-LAMP) for quantitative Salmonella Typhimurium (ST) detection. Quantum dot nanobeads (QBs) served as fluorescence reporters, providing good detection efficiency. The customizable strand displacement (SD) probe was used in LAMP to improve the specificity of the method and prevent by-product capture. Detection was based on a sandwich immunoassay. A fluorescence strip reader measured the fluorescence intensity (FI) of the test (T) line and control (C) line. The linear detection range of the strip was 102–108 colony forming units (CFU)·mL−1. The visual limit of detection was 103 CFU·mL−1, indicating that the system was ten-fold more sensitive than AuNPs-labelled test strips. ST specificity was analyzed in accordance with agarose gel outputs of polymerase chain reaction (PCR) and SD-LAMP. We detected ST in foods with an acceptable recovery of 85%–110%. The method is rapid, simple, almost equipment-free, and suitable for bacterial detection in foods and for clinical diagnosis.

ABSTRACT: Objectives: The aim of this study was to investigate the prevalence and genetic environment of the multidrug resistance gene lsa(E) in food-related Staphyloccocus aureus in China. Methods: 1463 S. aureus from retail food products in 39 Chinese cities were investigated to determine the prevalence of lsa(E). Furthermore, antimicrobial susceptibility testing, whole-genome sequencing (WGS), and complete genetic analysis were performed in lsa(E)-positive isolates. Results: Thirty-five isolates (2.4%) were positive for the lsa(E) gene, which had an extensive multidrug-resistance phenotype. ST9-t899 and ST1-t4792 were the common sequence types in positive strains. The lsa(E) genes were located in two different types of novel multidrug resistance region (MRRlsa[E]) on the chromosome. The Sa-MRRlsa(E)-I were inserted into the lctP gene. The Sa-MRRlsa(E)-II were inserted into the crtP gene, and were comprised of seven antibiotic resistance genes (ARGs) interspersed with varieties of insertion sequences (ISs), transposons, and DNA invertase genes, showing a novel arrangement harboring lsa(E). Part of transposon Tn1546 was inserted downstream of lnu(B) in the novel Sa-MRRlsa(E)-II. Both types of Sa-MRRlsa(E) could be excised from the chromosome, indicating that Sa-MRRlsa(E) may be transferable. Conclusion: Our study is the first systematical investigation of lsa(E)-positive S. aureus in retail foods in China. It indicated that the origin of most food-related lsa(E)-positive S. aureus in China might be associated with livestock or poultry breeding farms, and these strains may be transmitted between animals and food. S. aureus carrying novel Sa-MRRlsa(E), especially, serve as reservoirs of antibiotic resistance traits, and warrant further attention.

Abstract The emergence of the novel GII.17 Kawasaki 2014 norovirus variant raising the interest of the public, has replaced GII.4 as the predominant cause of noroviruses outbreaks in East Asia during 2014–2015. Antigenic variation of the capsid protein is considered as one of the key mechanisms of norovirus evolution. In this study, we screened a panel of GII.17 mutants. First, we produced norovirus P proteins using cell-free protein synthesis (CFPS) system, comparing the results to pure proteins expressed in a cell-based system. Next, we determined the binding capability of specific monoclonal antibody (mAb) 2D11 using a unique set of wild-type GII.17 strains. Results of the EIA involving a panel of mutant cell-free proteins indicated that Q298 was the key residue within loop 1. These data highlighted the essential residues in the linear antibody binding characteristics of novel GII.17. Furthermore, it supported the CFPS as a promising tool for rapidly screening mutants via the scalable expression of norovirus P proteins.

Filter-feeding oyster is an important food transmission vector for the foodborne norovirus. To investigate the contamination level and genetic diversity of norovirus in commercial oysters in Guangzhou and better assess the risk of consumption, oyster samples were collected from local aquatic market from June 2020 to May 2021 in this study. The contamination content and genotype distribution of virus in shellfish were detected by protease K treatment coupled with polyethylene glycol precipitation small system previously established in the laboratory，including the real-time reverse transcription polymerase chain reaction and the nested RT-PCR. 110 oyster samples were detected, the positive rate of norovirus genogroup II(GII) was 52.7% (58/110), and the contents of virus was 1.56×103~1.09×106 copies/g (digestive gland). The positive rate of norovirus in oysters collected in spring and summer (March to August) was 35.7% (20/56), which was lower than the positive rate of 70.4% (38/54) in autumn and winter (September to February). However, there was no significant difference in content of virus detected in all seasons, in spring, content of virus was (2.69±1.46)×105 copies/g (digestive gland), in summer, content of virus was (1.97±2.16)×105 copies/g (digestive gland), in autumn, content of virus was (6.91±6.16)×104 copies/g (digestive gland), and winter was (4.83±2.99)×104 copies/g (digestive gland). Positive samples were sequenced and analyzed, the result showed that except for one was GII.17 genotype, the rest were all GII.4 genotypes (n=13), which was consistent with the local clinical epidemic genotypes. The results of this study showed that the level of norovirus contamination in commercial oysters of Guangzhou was severe, it is necessary to strengthen control work of prevention for virus, especially to remind consumers to fully process when eating oysters.

This study explored the prevalence of Cronobacter spp. in wet rice and flour products from Guangdong province, China, the molecular characteristics and antimicrobial susceptibility profiles of the isolates were identified. Among 249 samples, 100 (40.16%) were positive for Cronobacter spp., including 77 wet rice and 23 wet flour products. Eleven serotypes were characterized among 136 isolates with C. sakazakii O2 (n = 32) predominating. Forty-nine MLST patterns were assigned, 15 of which were new. C. sakazakii ST4 (n = 17) was the dominant ST, which is previously reported to have caused three deaths; followed by C. malonaticus ST7 (n = 15), which is connected to adult infections. All strains presented susceptibility to ampicillin/sulbactam, imipenem, aztreonam and trimethoprim/sulfamethoxazole. The isolates showed maximum resistance to cephalothin, and the resistance and intermediate rates were 91.91% and 3.68%, each. Two strains, croM234A1 and croM283-1, displayed resistance to three antibiotics. High contamination level and predominant number of pathogenic STs of Cronobacter in wet rice and flour products implied a potential risk to public healthiness. This survey could provide comprehensive information for establishing more targeted control methods for Cronobacter spp.

PurposeTo evaluate the association of gut microbiome signature and disease progression in locally advanced non-small cell lung cancer (LA-NSCLC) patients treated with concurrent chemoradiotherapy (CCRT) by fecal metagenome analysis.MethodsMetagenome-wide association studies on baseline fecal samples from 18 LA-NSCLC patients before CCRT and 13 controls from healthy first-degree relatives were performed. Among the 18 LA-NSCLC patients, six patients were defined as the long progression-free survival (long-PFS) group (PFS≥11 months) while another 12 were in the short-PFS group (PFS

Bacillus cereus, an important foodborne pathogen, poses a risk to food safety and quality. Robust biofilm formation ability is one of the key properties that is responsible for the food contamination and food poisoning caused by B. cereus, especially the emetic strains. To investigate the mechanism of biofilm formation in emetic B. cereus strains, we screened for the mutants that fail to form biofilms by using random mutagenesis toward B. cereus 892-1, an emetic strain with strong biofilm formation ability. When knocking out flgE, a flagellar hook encoding gene, the mutant showed disappearance of flagellar structure and swimming ability. Further analysis revealed that both pellicle and ring presented defects in the null mutant compared with the wild-type and complementary strains. Compared with the flagellar paralytic strains ΔmotA and ΔmotB, the inhibition of biofilm formation by ΔflgE is not only caused by the inhibition of motility. Interestingly, ΔflgE also decreased the synthesis of cereulide. To our knowledge, this is the first report showing that a flagellar component can both affect the biofilm formation and cereulide production in emetic B. cereus, which can be used as the target to control the biohazard of emetic B. cereus.

Mining novel specific molecular targets and establishing efficient identification methods are significant for detecting Pseudomonas aeruginosa, which can enable P. aeruginosa tracing in food and water. Pangenome analysis was used to analyze the whole genomic sequences of 2017 strains (including 1,000 P. aeruginosa strains and 1,017 other common foodborne pathogen strains) downloaded from gene databases to obtain novel species-specific genes, yielding a total of 11 such genes. Four novel target genes, UCBPP-PA14_00095, UCBPP-PA14_03237, UCBPP-PA14_04976, and UCBPP-PA14_03627, were selected for use, which had 100% coverage in the target strain and were not present in nontarget bacteria. PCR primers (PA1, PA2, PA3, and PA4) and qPCR primers (PA12, PA13, PA14, and PA15) were designed based on these target genes to establish detection methods. For the PCR primer set, the minimum detection limit for DNA was 65.4 fg/μl, which was observed for primer set PA2 of the UCBPP-PA14_03237 gene. The detection limit in pure culture without pre-enrichment was 105 colony-forming units (CFU)/ml for primer set PA1, 103 CFU/ml for primer set PA2, and 104 CFU/ml for primer set PA3 and primer set PA4. Then, qPCR standard curves were established based on the novel species-specific targets. The standard curves showed perfect linear correlations, with R2 values of 0.9901 for primer set PA12, 0.9915 for primer set PA13, 0.9924 for primer set PA14, and 0.9935 for primer set PA15. The minimum detection limit of the real-time PCR (qPCR) assay was 102 CFU/ml for pure cultures of P. aeruginosa. Compared with the endpoint PCR and traditional culture methods, the qPCR assay was more sensitive by one or two orders of magnitude. The feasibility of these methods was satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 29 ready-to-eat vegetable samples and was almost consistent with that of the national standard detection method. The developed assays can be applied for rapid screening and detection of pathogenic P. aeruginosa, providing accurate results to inform effective monitoring measures in order to improve microbiological safety.

Although lactic acid bacteria (LAB) were shown to be effective for preventing photoaging, the underlying molecular mechanisms have not been fully elucidated. Accordingly, we examined the anti-photoaging potential of 206 LAB isolates and discovered 32 strains with protective activities against UV-induced injury. All of these 32 LABs exhibited high levels of 2,2-diphenyl-picrylhydrazyl, as well as hydroxyl free radical scavenging ability (46.89–85.13% and 44.29–95.97%, respectively). Genome mining and metabonomic verification of the most effective strain, Limosilactobacillus fermentum XJC60, revealed that the anti-photoaging metabolite of LAB was nicotinamide (NAM; 18.50 mg/L in the cell-free serum of XJC60). Further analysis revealed that LAB-derived NAM could reduce reactive oxygen species levels by 70%, stabilize the mitochondrial membrane potential, and increase the NAD+/NADH ratio in UV-injured skin cells. Furthermore, LAB-derived NAM downregulated the transcript levels of matrix metalloproteinase (MMP)-1, MMP-3, interleukin (IL)-1β, IL-6, and IL-8 in skin cells. In vivo, XJC60 relieved imflammation and protected skin collagen fiber integrity in UV-injured Guinea pigs. Overall, our findings elucidate that LAB-derived NAM might protect skin from photoaging by stabilizing mitochondrial function, establishing a therotical foundation for the use of probiotics in the maintenance of skin health.

Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF) spectrometry fingerprinting has reduced turnaround times, costs, and labor as conventional procedures in various laboratories. However, some species strains with high genetic correlation have not been directly distinguished using conventional standard procedures. Metabolomes can identify these strains by amplifying the minor differences because they are directly related to the phenotype. The pseudotargeted metabolomics method has the advantages of both non-targeted and targeted metabolomics. It can provide a new semi-quantitative fingerprinting with high coverage. We combined this pseudotargeted metabolomic fingerprinting with deep learning technology for the identification and visualization of the pathogen. A variational autoencoder framework was performed to identify and classify pathogenic bacteria and achieve their visualization, with prediction accuracy exceeding 99%. Therefore, this technology will be a powerful tool for rapidly and accurately identifying pathogens.

Although noroviruses are the causative agents of most non-bacterial foodborne disease outbreaks, effective antivirals are currently unavailable. Certain probiotic strains have been reported as active antivirals for norovirus infections, but their mechanisms have not been fully elucidated. Herein, we examined the antiviral potential of 122 lactic acid bacteria isolates against murine norovirus (MNV), a human norovirus surrogate. A centenarian gut-derived strain, Limosilactobacillus fermentum PV22, exhibited the strongest MNV antagonism and reduced the viral titer by 2.23 ± 0.38 (log-value) in 5 min with stable activity at 25°C (P < 0.01). Genome mining revealed that its antiviral activity can be attributed to the synthesis of γ-aminobutyric acid, and this finding was experimentally verified. Furthermore, we demonstrated the safety of the isolate and its high intestinal colonization ability. In conclusion, we discovered a centenarian gut-derived L. fermentum strain with strong anti-norovirus activity and identified its antiviral metabolite. Our results will offer new solutions for the prevention and treatment of food-related norovirus infections.

The contamination of antibiotic resistance genes (ARGs) may directly threaten human health. This study used a metagenomic approach to investigate the ARG profile in a drinking water treatment system (DWTS) in south China. In total, 317 ARG subtypes were detected; specifically, genes encoding bacitracin, multidrug, and sulfonamide were widely detected in the DWTS. Putative ARG hosts included Acidovorax (6.0%), Polynucleobacter (4.3%), Pseudomonas (3.4%), Escherichia (1.7%), and Klebsiella (1.5%) as the enriched biomarkers in the DWTS, which mainly carried bacitracin, beta-lactam, and aminoglycoside ARGs. From a further analysis of ARG-carrying contigs (ACCs), Stenotrophomonas maltophilia and Pseudomonas aeruginosa were the most common pathogens among the 49 ACC pathogens in the DWTS. The metagenomic binning results demonstrated that 33 high-quality metagenome-assembled genomes (MAGs) were discovered in the DWTS; particularly, the MAG identified as S. maltophilia-like (bin.195) harbored the greatest number of ARG subtypes (n = 8), namely, multidrug (n = 6; smeD, semE, multidrug_transporter, mexE, semB, and smeC), beta-lactam (n = 1; metallo-beta-lactamase), and aminoglycoside [n = 1; aph(3’)-IIb]. The strong positive correlation between MGEs and ARG subtypes revealed a high ARG dissemination risk in the DWTS. Based on the pure-culture method, 93 isolates that belong to 30 genera were recovered from the DWTS. Specifically, multidrug-resistant pathogens and opportunistic pathogens such as P. aeruginosa, Bacillus cereus, and S. maltophilia were detected in the DWTS. These insights into the DWTS’s antibiotic resistome indicated the need for more comprehensive ARG monitoring and management in the DWTS. Furthermore, more effective disinfection methods need to be developed to remove ARGs in DWTSs, and these findings could assist governing bodies in the surveillance of antibiotic resistance in DWTSs.

The commensal microbiome influences skin immunity, but its function in toenail health remains unclear. Paronychia is one of the most common inflammatory toenail diseases, but antibiotic treatment is seldom effective in clinical cases. In this study, we performed 16S rRNA sequencing to investigate the characteristics of microbes associated with paronychia in order to identify the key microorganisms involved in inflammation. Seventy dermic samples were collected from patients with paronychia and the differences in dermic microbiota were analyzed in patients with different inflammation severities. Distinct clustering of dermal microbiota was observed in the dermis with different inflammation severities. A higher relative abundance of anaerobic microorganisms such as Parvimona, Prevotella, and Peptoniphilus was observed in severe paronychia, whereas Lactobacillus disappeared with disease progression. Co-occurring network analysis suggested that the disturbance of the dermic microbiome and attenuation of antagonism by Lactobacillus against anaerobic pathogens may aggravate inflammation in paronychia. Functional analysis showed that dermic microbiome disturbance may worsen microbial metabolism and tissue repair in the skin. In conclusion, we revealed that an increased abundance of anaerobic microorganisms and loss of Lactobacillus in the dermis may promote paronychia progression and microbiological imbalance may aggravate inflammation in patients with paronychia.

Currently, invasive infections caused by Staphylococcus argenteus, which is a recently named staphylococcal species, are increasingly reported worldwide. However, only a few genomic studies of S. argenteus have offered comprehensive information regarding its genetic diversity, epidemiological characteristics, antimicrobial resistance genes (ARGs), virulence genes and other profiles. Here, we describe a comparative genomic analysis by population structure, pangenome, panmobilome, region-specific accessory genes confer an adaptive advantage in 153 S. argenteus strains which comprised 24 strains sequenced in this study and 129 strains whose genome sequences were available from GenBank. As a result, the population of S. argenteus comprised seven genetically distinct clades, including two major clades (C1 and C2), with distinct isolation source patterns. Pangenome analysis revealed that S. argenteus has an open pangenome composed of 7,319 genes and a core genome composed of 1,508 genes. We further determined the distributions of 75 virulence factors (VFs) and 30 known ARGs and identified at least four types of plasmids and 93 complete or partial putative prophages. It indicate that S. argenteus may show a similar level of pathogenicity to that of S. aureus. This study also provides insights into the evolutionary divergence of this pathogen, indicating that the geographical distribution was a potential driving force behind the evolutionary divergence of S. argenteus. The preferential horizontal acquisition of particular elements, such as staphylococcal cassette chromosome mec elements and plasmids, was observed in specific regions, revealing potential gene exchange between S. argenteus strains and local S. aureus strains. Moreover, multiple specific genes related to environmental adaptation were identified in strains isolated from East Asia. However, these findings may help promote our understanding of the evolutionary divergence of this bacterium at a high genetic resolution by providing insights into the epidemiology of S. argenteus and may help combat its spread.

Foodbone norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Candidate vaccines are being developed, however, no licensed vaccines are currently available for managing NoV infections. Screening for stimulated antibodies with broad-spectrum binding activities can be performed for the development of NoV polyvalent vaccines. In this study, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for testing the broad spectrum of anti-NoV antibodies. Capsid P proteins from 28 representative NoV strains (GI.1–GI.9 and GII.1–GII.22 except GII.11, GII.18, and GII.19) were selected, prepared, and used as coating antigens on one microplate. Combined with incubation and the horseradish peroxidase chromogenic reaction, the entire process for testing the spectrum of unknown antibodies required 2 h for completion. The intra-assay and inter-assay coefficients of variation were less than 10%. The new method was successfully performed with monoclonal antibodies and polyclonal antibodies induced by multiple antigens. In conclusion, the indirect ELISA assay developed in this study had a good performance of reliability, convenience, and high-throughput screening for broad-spectrum antibodies.

Small non-coding RNAs (sRNAs) in bacteria are important regulatory molecules for controlling virulence. In Vibrio spp., Qrr sRNAs are critical for quorum-sensing pathways and regulating the release of some virulence factors. However, the detailed role of Qrr sRNAs in the virulence of Vibrio parahaemolyticus remains poorly understood. In this study, we identified a Vibrio sRNA Qrr5 that positively regulates cytotoxicity and adherence in Caco-2 cells by primarily regulating the T3SS1 gene cluster. A number of 185, 586, 355, and 74 differentially expressed genes (DEGs) detected at 0, 2, 4, and 6 h post-infection, respectively, were mainly associated with ABC transporters and two-component system pathways. The DEGs exhibited a dynamic change in expression at various time points post-infection owing to the deletion of Qrr5. Accordingly, 17 related genes were identified in the co-expression network, and their interaction with Qrr5 was determined based on weighted co-expression network analysis during infection. Taken together, our results provide a comprehensive transcriptome profile of V. parahaemolyticus during infection in Caco-2 cells.

Background: Fermented milk is beneficial for metabolic disorders, while the underlying mechanisms of action remain unclear. This study explored the benefits and underlying mechanisms of Bifidobacterium longum 070103 fermented milk (BLFM) in thirteen-week high-fat and high-sugar (HFHS) fed mice using omics techniques. Methods and results: BLFM with activated glucokinase (GK) was screened by a double-enzyme coupling method. After supplementing BLFM with 10 mL/kg BW per day, fasting blood glucose, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and leptin were significantly reduced compared with the HFHS group. Among them, the final body weight (BW), epididymal fat, perirenal fat, and brown fat in BLFM group had better change trends than Lacticaseibacillus rhamnosus GG fermented milk (LGGFM) group. The amplicon and metabolomic data analysis identified Bifibacterium as a key gut microbiota at regulating glycolipid metabolism. BLFM reverses HFHS-induced reduction in bifidobacteria abundance. Further studies showed that BLFM significantly reduces the content of 3-indoxyl sulofphate associated with intestinal barrier damage. In addition, mice treated with BLFM improved BW, glucose tolerance, insulin resistance, and hepatic steatosis. Conclusion: BLFM consumption attenuates obesity and related symptoms in HFHS-fed mice probably via the modulation of gut microbes and metabolites.

The process of soybean fermentation has been practiced for more than 3,000 years. Although Dajiang and Sufu are two popular fermented soybean products consumed in North China, limited information is available regarding their microbial composition. Hence, the current study sought to investigate, and compare, the physicochemical indicators and microbial communities of traditional Dajiang and Sufu. Results showed that the titratable acidity (TA), and salinity, as well as the lactic acid, and malic acid contents were significantly higher in Sufu samples compared to Dajiang. Furthermore, Sufu samples contain abundant sucrose and fructose, while the acetic acid content was lower in Sufu compared to Dajiang samples. Moreover, the predominant bacterial phyla in Dajiang and Sufu samples were Firmicutes and Proteobacteria, while the major genera comprise Bacillus, Lactobacillus, Tetragenococcus, and Weissella. Moreover, Dajiang samples also contained abundant Pseudomonas, and Brevundimonas spp., while Halomonas, Staphylococcus, Lysinibacillus, Enterobacter, Streptococcus, Acinetobacter, and Halanaerobium spp. were abundant in Sufu samples. At the species level, Bacillus velezensis, Tetragenococcus halophilus, Lactobacillus rennini, Weissella cibaria, Weissella viridescens, Pseudomonas brenneri, and Lactobacillus acidipiscis represented the major species in Dajiang, while Halomonas sp., Staphylococcus equorum, and Halanaerobium praevalens were the predominant species in Sufu. Acetic acid and sucrose were found to be the primary major physicochemical factor influencing the bacterial communities in Dajiang and Sufu, respectively. Furthermore, Bacillus subtilis is strongly correlated with lactic acid levels, L. acidipiscis is positively correlated with acetic acid levels, while Staphylococcus sciuri and S. equorum are strongly, and positively, correlated with malic acid. Following analysis of carbohydrate and amino acid metabolism in all samples, cysteine and methionine metabolism, as well as fatty acid biosynthesis-related genes are upregulated in Dajiang compared to Sufu samples. However, such as the Staphylococcus, W. viridescens, and P. brenneri, as potentially foodborne pathogens, existed in Dajang and Sufu samples. Cumulatively, these results suggested that Dajiang and Sufu have unique bacterial communities that influence their specific characteristics. Hence, the current study provides insights into the microbial community composition in Dajiang and Sufu samples, which may facilitate the isolation of functional bacterial species suitable for Dajiang and Sufu production, thus improving their production efficiency.

Phenol is a toxic organic molecule that is widely detected in the natural environment, even in drinking water sources. Biological methods were considered to be a good tool for phenol removal, especially microbial immobilized technology. However, research on the “seed” bacteria along with microbial community analysis in oligotrophic environment such as drinking water system has not been addressed. In this study, Acinetobacter sp. DW-1 with high phenol degradation ability had been isolated from a drinking water biofilter was used as seeded bacteria to treat phenol micro-polluted drinking water source. Meanwhile, the whole genome of strain DW-1 was sequenced using nanopore technology. The genomic analysis suggests that Acinetobacter sp. DW-1 could utilize phenol via the β-ketoadipate pathway, including the catechol and protocatechuate branches. Subsequently, a bio-enhanced polyhedral hollow polypropylene sphere (BEPHPS) filter was constructed to investigate the stability of the seeded bacteria during the water treatment process. The denatured gradient gel electrophoresis (DGGE) profile and the quantification of phenol hydroxylase gene results indicate that when the BEPHPS filter was operated for 56 days, Acinetobacter sp. was still a persistent and competitive bacterium in the treatment group. In addition, 16S rRNA gene amplicon sequencing results indicate that Acinetobacter sp., as well as Pseudomonas sp., Nitrospira sp., Rubrivivax sp. were the predominant bacteria in the treatment group, which were different from that in the CK group. This study provides a better understanding of the mechanisms of phenol degradation by Acinetobacter sp. DW-1 at the gene level, and provides new insights into the stability of seeded bacteria and its effects on microbial ecology during drinking water treatment.

Human norovirus is a common cause of acute gastroenteritis worldwide, and oysters have been found to be the main carriers for its spread. The lack of efficient pre-treatment methods has been a major bottleneck limiting the detection of viruses in oysters. In this study, we established a novel immunomagnetic enrichment method using polyamidoamine (PAMAM) dendrimer/SA-biotin-mediated cascade amplification for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. We compared the capture efficiency of traditional immunomagnetic enrichment, biotin-amplified immunomagnetic enrichment, and PAMAM dendrimer/SA-biotin-mediated cascade-amplification immunomagnetic enrichment. The optimal capture efficiency of the novel method was 44.26 ± 1.45%, which increased by 183.17% (P < 0.01) and 18.09% (P < 0.05) compared with the first two methods, respectively. Three methods were all applied in detecting norovirus in 44 retail oysters, the detection rate of the PAMAM dendrimer/SA-biotin-mediated method was 25.0%, which was higher than those of traditional IME (15.90%) and SA-biotin-amplified IME (18.80%) by 9.1 and 6.2%, respectively. In conclusion, the novel method can be applied for the rapid detection of norovirus in oysters, which can help reduce the cost and time of detection and improve detection rates.

Vibrio parahaemolyticus is a common foodborne pathogen that causes gastroenteritis worldwide. Determining its prevalence and genetic diversity will minimize the risk of infection and the associated economic burden. Multilocus sequence typing (MLST) is an important tool for molecular epidemiology and population genetic studies of bacteria. Here, we analyzed the genetic and evolutionary relationships of 162 V. parahaemolyticus strains isolated in the Guangdong Province, China, using MLST. In the study, 120 strains were isolated from food samples, and 42 strains were isolated from clinical samples. All strains were categorized into 100 sequence types (STs), of which 58 were novel (48 from the food isolates and 10 from the clinical isolates). ST415 was the most prevalent ST among the food isolates, while ST3 was the most prevalent ST among the clinical isolates. Further, 12 clonal complexes, 14 doublets, and 73 singletons were identified in all ST clusters, indicating high genetic diversity of the analyzed strains. At the concatenated sequence level, non-synonymous sites in both, food and clinical isolates, were associated with purifying selection. Of note, the dN/dS ration was greater than 1 for some housekeeping genes in all isolates. This is the first time that some loci under positive selection were identified. These observations confirm frequent recombination events in V. parahaemolyticus. Recombination was much more important than mutation for genetic heterogeneity of the food isolates, but the probabilities of recombination and mutations were almost equal for the clinical isolates. Based on the phylogenetic analysis, the clinical isolates were concentrated in the maximum-likelihood tree, while the food isolates were heterogeneously distributed. In conclusion, the food and clinical isolates of V. parahaemolyticus from the Guangdong Province are similar, but show different evolutionary trends. This may help prevent large-scale spread of highly virulent strains and provides a genetic basis for the discovery of microevolutionary relationships in V. parahaemolyticus populations.

Salmonella is a widely distributed foodborne pathogen. The use of Salmonella phages as biocontrol agents has recently gained significant interest. Because the Salmonella genus has high diversity, efforts are necessary to identify lytic Salmonella phages focusing on different serovars. Here, five Salmonella phages were isolated from soil samples, and vB_SalP_TR2 was selected as a novel phage with high lytic potential against the host Salmonella serovar Albany, as well as other tested serovars, including Corvallis, Newport, Kottbus, and Istanbul. Morphological analyses demonstrated that phage vB_SalP_TR2 belongs to the Podoviridae family, with an icosahedral head (62 ± 0.5 nm in diameter and 60 ± 1 nm in length) and a short tail (35 ± 1 nm in length). The latent period and burst size of phage vB_SalP_TR2 was 15 min and 211 PFU/cell, respectively. It contained a linear dsDNA of 71,453 bp, and G + C content was 40.64%. Among 96 putative open reading frames detected, only 35 gene products were found in database searches, with no virulence or antibiotic resistance genes being identified. As a biological control agent, phage vB_SalP_TR2 exhibited a high temperature and pH tolerance. In vitro, it lysed most S. Albany after 24 h at 37°C with multiplicities of infection of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100. In food matrices (milk and chicken meat), treatment with phage vB_SalP_TR2 also reduced the number of S. Albany compared with that in controls. These findings highlighted phage vB_SalP_TR2 as a potential antibacterial agent for the control of Salmonella in food samples.

Abstract Background Human norovirus is regarded as the leading cause of nonbacterial acute diarrhea in developing and developed countries. Among all genotypes, GII.4 has been the predominant genotype, but in East Asia, it was replaced by the GII.17 in 2014/2015. However, after the prevalence of new GII.17 variant in South China, a sharply increase in the number of norovirus infections associated with sporadic acute diarrhea was detected. In this study, we would investigate the prevalence and genetic diversity of noroviruses in the sporadic acute gastroenteritis cases in the post-GII.17 period in South China. Methods Norovirus was screened from 217 patients with sporadic acute gastroenteritis from August 2015 to October 2017 by reverse transcription-polymerase chain reaction. Then, two regions including the partial RNA polymerase and the capsid gene of positive samples were amplified and sequenced. Phylogenetic analyses were performed to determine norovirus genotypes. Complete VP1 sequences of GII.4 strains detected in this study were also amplified and subjected into evolutionary tracing analyses. Results A total of 43 (19.82%) norovirus samples were confirmed from 217 stool specimens, and it was found that GII.4 resurged as the new predominant variant, accounting for 76.74% (33/43) of positive samples. Only one local strain GZ2015-L550 was clustered with the contemporary GII.P16/GII.4–2012 recombinant variant, and other 32 local strains belonged to the clade with the GII.Pe/GII.4–2012 variant. Other genotypes including GII.17 (n = 4), GII.3 (n = 4), GII.8 (n = 1) and GI. 6 (n = 1) were also detected. Furthermore, all GII.4 strains were phylogenetic analyzed based on their capsid P2 subdomains. Combined with other reported 754 strains, the GII.4–2012 variant could be divided into two clades. Most GII.4 strains collected in 2016 and 2017 in this study (7/8) formed a new cluster A in Clade II with additional 103 contemporaneous strains. In addition, evolutionary tracing of the capsid P2 subdomain of this variant was also analyzed, and one specific amino acid substitutions (N373) was identified for Cluster A. Conclusion In summary, this study confirmed a norovirus infection peak in the post-GII.17 period in South China, which was caused by the resurgence of the GII.4 variant.

Abstract Background Ready-to-eat (RTE) vegetables have become increasingly popular along with the trend of moving towards a healthy lifestyle. However, RTE vegetables are at a higher risk of containing pathogens, maybe owing to lack of rigorous sanitization procedures. To understand the prevalence and potential risk of Listeria monocytogenes in RTE vegetables, we investigated the contamination level and characteristics of L. monocytogenes isolated from fresh vegetables. Results Twenty-three (5.49%) of the 419 vegetables samples were positive for L. monocytogenes. Phylogenetic group I.1 (1/2a-3a) and II.2 (1/2b-3b-7) strains were predominant in 30 isolates, which accounted for 33.3 and 50.0%, respectively. Multilocus sequence typing of the 30 isolates grouped them into nine sequence types (STs). The most common STs were ST87 (36.7%) and ST8 (26.7%). Virulence analysis showed that all 30 isolates harbored eight classical virulence genes, 10.0% isolates harbored the llsX gene (ST3 and ST1 strains), and 36.7% carried the ptsA gene and belonged to ST87. Approximately 83.3% isolates carried full-length inlA, whereas five isolates had premature stop codons in inlA, three of which belonged to ST9 and two to ST8. Antibiotic susceptibility showed the isolates were varyingly resistant to 13 antibiotics, 26.7% of the isolates were multi-drug resistant. Conclusions The fresh vegetables contain some potential hypervirulent L. monocytogenes (ST1 and ST87) in the Chinese markets. In addition, the high rate of L. monocytogenes isolates was multi-drug resistant. Fresh raw vegetables may be a possible transmission route for L. monocytogenes infection in consumers. Therefore, sanitization of raw fresh vegetables should be strengthened to ensure their microbiological safety when used as RTE vegetables.

Probiotic fermented milk can lower the incidence rate of hypertension and is beneficial to the regulation of the intestinal microecology. However, the underlying molecular mechanism remains elusive. Here, we evaluated the role of the gut microbiota and its metabolites in the antihypertensive effect of milk fermented by the Lactiplantibacillus plantarum strains SR37-3 (PFM-SR37-3) and SR61-2 (PFM-SR61-2) in Ng-nitro-L-arginine methyl ester induced hypertensive rats. The results showed that PFM-SR37-3 and PFM-SR61-2 intervention significantly lowered the blood pressure (BP) of NG-nitro-L-arginine methyl ester induced hypertensive rats and attenuated renal injury. In particular, long-term administration of PFM inhibited a progressive elevation in SBP (170.22 ± 8.40 and 133.28 ± 6.09 by model group and PFM-SR37-3 treated model group, respectively, at the end of the 4 weeks; p < 0.01 PFM-SR37-3 treated model group versus model group) and DBP (133.83 ± 5.91 and 103.00 ± 6.41 by model group and PFM-SR37-3 treated model group, respectively, at the end of the 4 weeks; p < 0.01 PFM-SR37-3 treated model group versus model group). PFM-SR37-3 and PFM-SR61-2 reshaped the gut microbiome and metabolome, and especially regulated the metabolic levels of L-phenylalanine, L-methionine and L-valine in the intestine and blood circulation. The analysis of the target organ’s aortic transcriptome indicated that the protective effects of PFM-SR37-3 and PFM-SR61-2 were accompanied by the modulation of the BP circadian rhythm pathway, which was conducive to cardiovascular function. Vascular transcriptomic analysis showed that circadian rhythm and AMPK might be potential targets of hypertension. In addition, the ACE inhibition rates of Lactiplantibacillus plantarum SR37-3 and Lactiplantibacillus plantarum SR61-2 in vitro were 70.5% and 68.9%, respectively. Our research provides new insights into novel and safe options for hypertension treatment.

Human norovirus is regarded as the leading cause of epidemic acute gastroenteritis with GII.4 being the predominant genotype during the past decades. In the winter of 2014/2015, the GII.17 Kawasaki 2014 emerged as the predominant genotype, surpassing GII.4 in several East Asian countries. Hence, the influence of host immunity response on the continuous evolution of different GII.17 variants needs to be studied in depth. Here, we relate the inferences of evolutionary mechanisms of different GII.17 variants with the investigation of cross-reactivity and cross-protection of their respective antisera using the expression of norovirus P particles in Escherichia coli. The cross-reactivity assay showed that the antisera of previous strains (GII.17 A and GII.17 B) reacted with recent variants (GII.17 C and GII.17 D) at high OD values from 0.8 to 1.16, while recent variant antisera cross-reacting with previous strains were weak with OD values between 0.26 and 0.56. The cross-protection assay indicated that the antisera of previous strains had no inhibitory effect on recent variants. Finally, mutations at amino acids 353–363, 373–384, 394–404, and 444–454 had the greatest impact on cross-reactivity. These data indicate that the recent pandemic variants GII.17 C and GII.17 D avoided the herd immunity effect of previous GII.17 A and GII.17 B strains through antigenic variation.

Vibrio parahaemolyticus is an important foodborne pathogen and its biofilm formation ability facilitates its colonization and persistence in foods by protecting it from stresses including environmental variation and antibiotic exposure. Several important proteins are involved in biofilm formation; however, the identity and function of many remain unknown. In this study, we discovered a hypothetical protein, VP0610 that negatively regulates biofilm formation in Vibrio parahaemolyticus, and we found that the loss of vp0610 typically results in pleiotropic phenotypes that contribute toward promoting biofilm formation, including significantly increased insoluble exopolysaccharide production and swimming motility, decreased soluble exopolysaccharide production, and decreased bis-(3′-5′)-cyclic dimeric guanosine monophosphate production. Pull-down assays revealed that VP0610 can interact with 180 proteins, some of which (Hfq, VP0710, VP0793, and CyaA) participate in biofilm formation. Moreover, deleting vp0610 enhanced the expression of genes responsible for biofilm component (flaE), the sugar phosphotransferase system (PTS) EIIA component (vp0710 and vp0793), and a high-density regulator of quorum sensing (opaR), while reducing the expression of the bis-(3′-5′)-cyclic dimeric guanosine monophosphate degradation protein (CdgC), resulting in faster biofilm formation. Taken together, our results indicate that vp0610 is an integral member of the key biofilm regulatory network of V. parahaemolyticus that functions as a repressor of biofilm formation.

The accurate and rapid classification of Salmonella serovars is an essential focus for the identification of isolates involved in disease in humans and animals. The purpose of current research was to identify novel sensitive and reliable serovar-specific targets and to develop PCR method for Salmonella C2 serogroups (O:8 epitopes) in food samples to facilitate timely treatment. A total of 575 genomic sequences of 16 target serovars belonging to serogroup C2 and 150 genomic sequences of non-target serovars were analysed by pan-genome analysis. As a result, four and three specific genes were found for serovars Albany and Hadar, respectively. Primer sets for PCR targeting these serovar-specific genes were designed and evaluated based on their specificity; the results showed high specificity (100%). The sensitivity of the specific PCR was 2.8 × 101–103 CFU/mL and 2.3 × 103–104 CFU/mL for serovars Albany and Hadar, respectively, and the detection limits were 1.04 × 103–104 CFU/g and 1.16 × 104–105 CFU/g in artificially contaminated raw pork samples. Furthermore, the potential functions of these serovar-specific genes were analysed; all of the genes were functionally unknown, except for one specific serovar Albany gene known to be a encoded secreted protein and one specific gene for serovars Hadar and Albany that is a encoded membrane protein. Thus, these findings demonstrate that pan-genome analysis is a precious method for mining new high-quality serovar-targets for PCR assays or other molecular methods that are highly sensitive and can be used for rapid detection of Salmonella serovars.

Antimicrobial resistance has become a major public health threat. Food-related Staphylococcus species have received much attention due to their multidrug resistance. The cfr gene associated with multidrug resistance has been consistently detected in food-derived Staphylococcus species. In this retrospective study, we examined the prevalence of cfr-positive Staphylococcus strains isolated from poultry meat in different geographical areas of China from 2011 to 2016. Two cfr-positive Staphylococcus delphini strains were identified from poultry meat in China. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in the two S. delphini isolates 245-1 and 2794-1. Whole-genome sequencing showed that they both harbored a novel 20,258-bp cfr-carrying Tn558 transposon derivative on their chromosomes. The Tn558 derivative harbors multiple antimicrobial resistance genes, including the transferable multiresistance gene cfr, chloramphenicol resistance gene fexA, aminoglycoside resistance genes aacA-aphD and aadD, and bleomycin resistance gene ble. Surprisingly, within the Tn558 derivative, an active unconventional circularizable structure containing various resistance genes and a copy of a direct repeat sequence was identified by two-step PCR. Furthermore, core genome phylogenetic analysis revealed that the cfr-positive S. delphini strains were most closely related to S. delphini 14S03313-1 isolated from Japan in 2017 and 14S03319-1 isolated from Switzerland in 2017. This study is the first report of S. delphini harboring a novel cfr-carrying Tn558 derivative isolated from retail food. This finding raises further concerns regarding the potential threat to food safety and public health safety. The occurrence and dissemination of similar cfr-carrying transposons from diverse Staphylococcus species need further surveillance.

Listeria spp. is an important foodborne disease agent, often found in the fresh mushroom (Flammulina velutipes) and its production environment. The aim of this study was to develop multiplex PCR for rapid identification of Listeria monocytogenes and Listeria ivanovii, and nonpathogenic Listeria in F. velutipes plants. Pan-genome analysis was first used to identify five novel Listeria-specific targets: one for the Listeria genus, one for L. monocytogenes, and three for L. ivanovii. Primers for the novel targets were highly specific in individual reactions. The detection limits were 103–104 CFU/mL, meeting the requirements of molecular detection. A mPCR assay for the identification of pathogenic Listeria, with primers targeting the novel genes specific for Listeria genus (LMOSLCC2755_0944), L. monocytogenes (LMOSLCC2755_0090), and L. ivanovii (queT_1) was then designed. The assay specificity was robustly verified by analyzing nonpathogenic Listeria and non-Listeria spp. strains. The determined detection limits were 2.0 × 103 CFU/mL for L. monocytogenes and 3.4 × 103 CFU/mL for L. ivanovii, for pure culture analysis. Further, the assay detected 7.6 × 104 to 7.6 × 100 CFU/10 g of pathogenic Listeria spiked into F. velutipes samples following 4–12 h enrichment. The assay feasibility was evaluated by comparing with a traditional culture-based method, by analyzing 129 samples collected from different F. velutipes plants. The prevalence of Listeria spp. and L. monocytogenes was 58.1% and 41.1%, respectively. The calculated κ factors for Listeria spp., L. monocytogenes, and L. ivanovii were 0.97, 0.97, and 1, respectively. The results of the novel mPCR assay were highly consistent with those of the culture-based method. The new assay thus will allow rapid, specific, and accurate detection and monitoring of pathogenic Listeria in food and its production environment.

Infection with Vibrio vulnificus is notorious for its atypical clinical manifestations and irreversible disease progression. Lysine acetylation is a conserved post-translational modification (PTM) that plays a critical regulatory role in diverse cellular processes. However, little is known about the role of lysine acetylation on the pathogenesis of V. vulnificus. Here, we report the complete genome sequence and a global profile for protein lysine acetylation of V. vulnificus Vv180806, a highly cefoxitin resistant strain isolated from a mortality case. The assembled genome comprised two circular chromosomes and one circular plasmid; it contained 4,770 protein-coding genes and 153 RNA genes. Phylogenetic analysis revealed genetic homology of this strain with other V. vulnificus strains from food sources. Of all the proteins in this strain, 1,924 (40.34%) were identified to be acetylated at 6,626 sites. The acetylated proteins were enriched in metabolic processes, binding functions, cytoplasm, and multiple central metabolic pathways. Moreover, the acetylation was found in most identified virulence factors of this strain, suggesting its potentially important role in bacterial virulence. Our work provides insights into the genomic and acetylomic features responsible for the virulence and antibiotic resistance of V. vulnificus, which will facilitate future investigations on the pathogenesis of this bacterium.

ABSTRACT Increasing data indicate that insects serve as major reservoirs and vectors of viruses, which account for the continuously increasing ecological burden and infectious disease outbreaks. Uncovering the hidden diversity of viruses in insects will further the understanding of the ecological and evolutionary perspectives in the emergence of insect-associated virus diseases. In this study, we queried transcriptome sequencing (RNA-Seq) data from more than 600 species across 32 insect orders dwelling in different ecological habitats and recovered more than 1,213 RNA viruses that were recapitulated in 40 families, 2 unclassified genera, and many unspecified viral groups. These novel viruses included the well-known insect-associated viruses within Flaviviridae, Picornavirales, Bunyavirales, Mononegavirales, Nidovirales, Reoviridae, and Negevirus. More appeared to form novel clusters within previously described taxa or could be resolved as paraphyletic, including the first astrovirus identified in insects, in which many were sufficiently divergent to warrant the establishment of new virus genera or families. Additionally, some viruses were closely related to the recognized plant-, fungus-, and vertebrate-specific species, implying the importance of relationships between insect behavior and virus spread. Comparative genome analyses also revealed high genomic variability with respect to the flexible gene pool and genome architecture of these newly described viruses, including the evidence for genome reshuffling first discovered in Dicistroviridae. The data reflecting the genetically and ecologically diverse viral populations in insects greatly expand our understanding of RNA viruses in nature and highlight that the biodiversity of RNA viruses remains largely unexplored. IMPORTANCE Insects comprise the largest proportion of animals on earth and are frequently implicated in the transmission of vector-borne diseases. However, considerable attention has been paid to the phytophagous and hematophagous insects, with results that provide insufficient and biased information about the viruses in insects. Here, we have delivered compelling evidence for the exceptional abundance and genetic diversity of RNA viruses in a wide range of insects. Novel viruses were found to cover major categories of RNA viruses, and many formed novel clusters divergent from the previously described taxa, dramatically broadening the range of known RNA viruses in insects. These newly characterized RNA viruses exhibited high levels of genomic plasticity in genome size, open reading frame (ORF) number, intergenic structure, and gene rearrangement and segmentation. This work provides comprehensive insight into the origin, spread, and evolution of RNA viruses. Of course, a large-scale virome project involving more organisms would provide more-detailed information about the virus infections in insects.

Vibrio parahaemolyticus is a marine and estuarine bacterium that leads to damage of aquatic industry by foodborne outbreaks and possesses an enormous threat to food safety as well as human health worldwide. In the current study, we investigated 905 food samples (ready-to-eat foods, fish, and shrimp) from 15 provinces in China, and aimed to determine prevalence, biological characteristics and genetic diversity of presumptive V. parahaemolyticus isolates. Firstly, 14.17% of 240 fish samples, 15.34% of 365 shrimp samples and 3.67% of 300 RTE food samples were positive for potential V. parahaemolyticus. Secondly, 69 food samples (14.87%) collected in summer were positive for target isolates, while the rate of positive sample of 441 food samples in winter reached 7.26%. Thirdly, we purified 202 V. parahaemolyticus strains for further research. And antimicrobial susceptibility results of strains tested revealed that the highest resistance rate was observed for ampicillin (79.20%). At the same time, 148 (73.27%) of all isolates were classified and defined as multi-drug resistant foodborne bacteria. The results of PCR assay showed that the isolates being positive for the tdh, trh or both genes, were up to 9.90%, 19.80% or 3.96%. Besides, multiplex PCR test showed that the isolates carrying O2 serogroup were the most prevalent. Furthermore, sequence types (STs) of 108 isolates were obtained via multi-locus sequence typing. Not only 82 STs were detected, but also 41 of which were updated in the MLST database. Thus, our findings significantly demonstrated the high contamination rates of V. parahaemolyticus in fish and shrimp and it may possess potential threat for consumer health. We also provided up-to-date dissemination of antibiotic-resistant V. parahaemolyticus which is important to ensure the high efficacy in the treatment of human and aquatic products infections. Lastly, with the identification of 82 STs including 41 novel STs, this study significantly revealed the high genetic diversity among V. parahaemolyticus. All of our research improved our understanding on microbiological risk assessment in ready-to-eat foods, fish, and shrimp.

Bacillus cereus is one of the most important foodborne pathogenic microorganisms, which can lead to gastrointestinal and non-gastrointestinal diseases. However, the potential risk of B. cereus in aquatic products in China has not been comprehensively evaluated yet. In this study, a total of 860 aquatic samples from three types of retail aquatic products were collected from 39 major cities in China from 2011 to 2016. The contamination, distribution of virulence genes, antibiotic resistance and genetic diversity of B. cereus isolates were measured and analyzed. Of all the samples, 219 (25.47%) were positive for B. cereus and 1.83% (4/219) of the samples had contamination levels of more than 1,100 most probable number (MPN)/g. Different isolates had virulence potential, within which 59.6% (164/275) contained all three kinds of enterotoxin genes (nhe, hbl, and cytK-2) and 5.1% (14/275) possessed cereulide encoding gene cesB. The antimicrobial resistance profiles revealed the universal antibiotic resistance to rifampin and most β-lactams, suggesting the necessity to continuously monitor the antibiotic resistance of B. cereus in aquatic products and to control drug use in aquaculture. In sum, our study indicates the potential hazards of B. cereus isolated from aquatic products to customers and may provide a reference for clinical treatment caused by B. cereus.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in humans. This study aimed to identify the pathogenic potential of L. monocytogenes isolated from ready-to-eat (RTE) foods and pasteurized milk in China on the basis of its phenotypic and genotypic characteristics. Approximately 7.7% (44/570) samples tested positive for L. monocytogenes among 10.8% (39/360) RTE and 2.4% (5/210) pasteurized milk samples, of which 77.3% (34/44) had < 10 MPN/g, 18.2% (8/44) had 10–110 MPN/g, and 4.5% (2/44) had > 110 MPN/g. A total of 48 strains (43 from RTE foods and five from milk samples) of L. monocytogenes were isolated from 44 positive samples. PCR-serogroup analysis revealed that the most prevalent serogroup was II.2 (1/2b-3b-7), accounting for 52.1% (25/48) of the total, followed by serogroup I.1 (1/2a-3a) accounting for 33.3% (16/48), serogroup I.2 (1/2c-3c) accounting for 12.5% (6/48), and serogroup II.1 (4b-4d-4e) accounting for 2.1%. All isolates were grouped into 11 sequence types (STs) belonging to 10 clonal complexes (CCs) and one singleton (ST619) via multi-locus sequence typing. The most prevalent ST was ST87 (29.2%), followed by ST8 (22.9%), and ST9 (12.5%). Virulence genes determination showed that all isolates harbored eight virulence genes belonging to Listeria pathogenicity islands 1 (LIPI-1) (prfA, actA, hly, mpl, plcA, plcB, and iap) and inlB. Approximately 85.4% isolates carried full-length inlA, whereas seven isolates had premature stop codons in inlA, six of which belonged to ST9 and one to ST5. Furthermore, LLS (encoded by llsX gene, representing LIPI-3) displays bactericidal activity and modifies the host microbiota during infection. LIPI-4 enhances neural and placental tropisms of L. monocytogenes. Results showed that six (12.5%) isolates harbored the llsX gene, and they belonged to ST1/CC1, ST3/CC3, and ST619. Approximately 31.3% (15/48) isolates (belonging to ST87/CC87 and ST619) harbored ptsA (representing LIPI-4), indicating the potential risk of this pathogen. Antimicrobial susceptibility tests revealed that > 95% isolates were susceptible to 16 antimicrobials; however, 60.4 and 22.9% isolates were intermediately resistant to streptomycin and ciprofloxacin, respectively. The results show that several isolates harbor LIPI-3 and LIPI-4 genes, which may be a possible transmission route for Listeria infections in consumers.

Sequence type 59 (ST59) is a predominant clonal lineage of community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) in Asia. Despite its increasing clinical relevance in China, the evolution and geographic expansion of ST59 has been relatively uncared for. Previous study has shown that ST59 was the predominant clone in food-related MRSA in China. This study compared the genomes of 87 clonal complex (CC) 59 S. aureus isolates sourced from food chain and infection cases to reconstruct the molecular evolution and geographical spread of ST59. Accordingly, three major sub-clades of ST59 were identified and these did not correlate with isolation source or location. Phylogenetic analysis estimated that ST59 in mainland China diverged from a most common recent ancestor around 1974, and most of the cases of cross-country transmission occurred between 1987 and 2000. Notably, two recent events of cross-country transmission through the food chain were observed, the isolates from these events diverged within relatively short time intervals. These isolates also showed high similarity in terms of their core genome, accessory genes, and antibiotic resistance patterns. These findings provide a valuable insight into the potential route of ST59 expansion in China and indicate a need for robust food chain surveillance to prevent the spread of this pathogen.

Campylobacter jejuni is a major foodborne pathogen worldwide. As it forms biofilms, it can become a persistent contaminant in the food and pharmaceutical industries. In this study, it was demonstrated that C. jejuni could make more biofilm in aerobic conditions than in microaerobic conditions, and only 13.9% C. jejuni entered coccus (a VBNC state) under microaerobic conditions; however, the rate increased to 95.5% under aerobic conditions. C. jejuni could form more biofilm in mixed culture with Escherichia coli or Pseudomonas aeruginosa than in pure culture. Scanning electron microscope results showed that C. jejuni retained its normal spiral shape under aerobic conditions for 48 h by forming crosslinks with the aerobic and facultative anaerobic bacteria. Additionally, culture medium containing 0.5 mg/ml ZnO nanoparticles inhibited biofilm formation. Our results provide information on a new approach to controlling contamination via C. jejuni.

Bacillus cereus is widely distributed in different food products and can cause a variety of symptoms associated with food poisoning. Since ready-to-eat (RTE) foods are not commonly sterilized by heat treatment before consumption, B. cereus contamination may cause severe food safety problems. In this study, we investigated the prevalence of B. cereus in RTE food samples from different regions of China and evaluated the levels of bacterial contamination, antibiotic resistance, virulence gene distribution, and genetic polymorphisms of these isolates. Of the tested retail RTE foods, 35% were positive for B. cereus, with 39 and 83% of the isolated strains harboring the enterotoxin-encoding hblACD and nheABC gene clusters, respectively. The entFM gene was detected in all B. cereus strains. The cytK gene was present in 68% of isolates, but only 7% harbored the emetic toxin-encoding gene cesB. Antimicrobial susceptibility testing revealed that the majority of the isolates were resistant not only to most β-lactam antibiotics, but also to rifamycin. Multilocus sequence typing (MLST) revealed that the 368 isolates belonged to 192 different sequence types (STs) including 93 new STs, the most prevalent of which was ST26. Collectively, our study indicates the prevalence, bacterial contamination levels, and biological characteristics of B. cereus isolated from RTE foods in China and demonstrates the potential hazards of B. cereus in RTE foods.

The aim of this study was to develop a potential microbial preservative to prevent the growth of fungi in food. The isolate ZC-G-5 showed strong antifungal activity against food spoilage fungi and Streptomyces albulus was identified on the basis of morphologic, culture, and 16S rDNA sequence analyses. The active metabolite was elucidated as tetramycin A (TMA) through spectroscopic techniques, including HR-ESI-MS, 1D-NMR, and 2D-NMR. An antifungal activity assay revealed that the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of TMA were 1.50–2.50 and 3.00–5.00 μg/ml, respectively. In situ antifungal activity analyses demonstrated that 90.0 μg/ml of TMA could inhibit the growth of fungi for over 14 days. In order to enhance TMA production, the high-yield mutant strain YB101 was screened, based on the isolate ZC-G-5, using a high-throughput screening method. The best metabolic precursor was selected during fermentation, when the concentration of glycerol was 8% (v/v) in Gauze’s broth medium to cultivate the mutant strain YB101; the concentration of TMA could be increased to 960.0 μg/ml, compared with the original isolate ZC-G-5, where the concentration of the TMA was only 225.0 μg/ml. Our study may contribute to the application of S. albulus and its active metabolite as a potential bio-preservative in the food industry.

Staphylococcus aureus is amongst the most virulent pathogens, causing chronic and life-threatening human infections. Methicillin-resistant S. aureus (MRSA) are multidrug-resistant strains, and the ability of forming a biofilm reduces their sensitivity to antibiotics. Thus, the alternative compounds inhibiting both resistant strains and biofilm formation are in high demand. In our study, the strain FJKB0103 was isolated from the rhizosphere of Garcinia mangostana, showing strong anti-MRSA activity. We performed molecular phylogenic analysis, analyzed average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), and biochemical characteristics to identify strain FJKB0103 as Pseudomonas protegens. Herein, the genome of strain FJKB0103 was sequenced and subjected to antiSMASH platform, mutational, and functional analyses. The FJKB0103 draft genome was 6,776,967 bp with a 63.4% G + C content, and 16 potential secondary compound biosynthetic clusters in P. protegens FJKB0103 were predicted. The deletion mutant and complementary analysis suggested that DAPG was the anti-MRSA compound. Further tests showed that MRSA strains were sensitive to DAPG, and the lysis of bacterial cells was observed at a high concentration of DAPG. Additionally, DAPG inhibited the biofilm formation of MRSA at subinhibitory concentration. These results suggested that DAPG might be a good alternative treatment to control infections caused by MRSA.

Vibrio parahaemolyticus causes aquatic vibriosis. Its biofilm protects it from antibiotics; therefore, a new different method is needed to control V. parahaemolyticus for food safety. Phage therapy represents an alternative strategy to control biofilms. In this study, the lytic Vibrio phage vB_VpaP_FE11 (FE11) was isolated from the sewers of Guangzhou Huangsha Aquatic Market. Electron microscopy analysis revealed that FE11 has a typical podovirus morphology. Its optimal stability temperature and pH range were found to be 20–50 °C and 5–10 °C, respectively. It was completely inactivated following ultraviolet irradiation for 20 min. Its latent period is 10 min and burst size is 37 plaque forming units/cell. Its double-stranded DNA genome is 43,397 bp long, with a G + C content of 49.24% and 50 predicted protein-coding genes. As a lytic phage, FE11 not only prevented the formation of biofilms but also could destroy the formed biofilms effectively. Overall, phage vB_VpaP_FE11 is a potential biological control agent against V. parahaemolyticus and the biofilm it produces.

This study investigated the molecular characteristics of rifampin-resistant (RIF-R) Staphylococcus aureus isolates recovered from 4300 retail food samples covering most provincial capitals in China, from 2011 to 2016. Of the 1463 S. aureus enrolled, 149 isolates (142 MSSA and 7 MRSA) were identified as rifampicin-resistant, including 20 high-level (MICs ≥ 8 μg/mL) and 129 low-level (MICs between 2 and 4 μg/mL) rifampicin-resistant strains. Most of the RIF-R S. aureus isolates were resistant to more than three antibiotics. The mutations in the rifampicin resistance-determining region of the rpoB gene were studied in all RIF-R strains. All of the strains presented the mutational change 481 His/Asn and five isolates presented an additional mutation, including 477 Asp/Tyr, 527 Ile/Met, and 466 Leu/Ser, respectively. Thirteen STs and twenty-one spa types were represented, in which five MRSA showed non-type SCCmec and the remaining MRSA belonged to SCCmec type IV—where, ST1-t127 was the predominant type from all of the isolates, while ST398-t034 was the predominant type for the MRSA isolates. In this study, we found that the food-related RIF-R S. aureus may have a unique genetic background selection. However, the scenario regarding the presence of RIF-R S. aureus, especially MRSA, in retail food in China is not favorable and warrants public attention.

The bacterial communities and physicochemical characteristics of four types of extremely distinctive traditionally fermented vegetables (pickled pepper (PP), pickled Brassica napobrassica (PBN), salted flowers of wild chives (SFWC), and pickled cucumber (PC)) were identified and compared from north China. Lactobacillus was the main bacterial genus in PP and PBN samples, with Oceanobacillus only being observed in PBN. The predominant genus in SFWC was Weissella, while in PC they were were Carnimonas and Salinivibrio. At the species level, Companilactobacillus ginsenosidimutans, Fructilactobacillus fructivorans, and Arcobacter marinus were abundant in PP and PBN. Levilactobacillus brevis and Companilactobacillus alimentarius were enriched in PP, and L. acetotolerans, Ligilactobacillus acidipiscis and Pediococcus parvulus were observed in PBN. Weissella cibaria and Kosakonia cowanii were abundant in SFWC. Moreover, tartaric acid was the most physicochemical factor influencing microbial composition, followed by malic acid, titratable acidity (TA), and lactic acid. Furthermore, functional analysis demonstrated that the most genes of the bacterial profiles correlated with carbohydrate metabolism. However, some foodborne pathogens were existed, such as Staphylococcus and Arcobacter marinus. The results of this study provide detailed insight into the relationship between the bacterial communities and physicochemical indices of fermented vegetables, and may improve the quality and safety of traditional Chinese fermented vegetables.