Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

Cold-tolerance mechanisms in foodborne pathogens: Escherichia coli and Listeria monocytogenes as examples.

The cold chain is an integral part of the modern food industry. Low temperatures can effectively alleviate food loss and the transmission of foodborne diseases caused by microbial reproduction. However, recent reports have highlighted shortcomings in the current cold chain technology's ability to prevent and control cold-tolerant foodborne pathogens. Furthermore, it has been observed that certain cold-chain foods have emerged as new sources of infection for foodborne disease outbreaks. Consequently, there is a pressing need to enhance control measures targeting cold-tolerant pathogens within the existing cold chain system. This paper aims to review the recent advancements in understanding the cold tolerance mechanisms of key model organisms, identify key issues in current research, and explore the potential of utilizing big data and omics technology in future studies.

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

A dual-recognition strategy is reported to construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and are robust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus. In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

GII.17[P17] and GII.8[P8] noroviruses showed different RdRp activities associated with their epidemic characteristics.

Norovirus is the primary foodborne pathogenic agent causing viral acute gastroenteritis. It possesses broad genetic diversity and the prevalence of different genotypes varies substantially. However, the differences in RNA-dependent RNA polymerase (RdRp) activity among different genotypes of noroviruses remain unclear. In this study, the molecular mechanism of RdRp activity difference between the epidemic strain GII.17[P17] and the non-epidemic strain GII.8[P8] was characterized. By evaluating the evolutionary history of RdRp sequences with Markov Chain Monte Carlo method, the evolution rate of GII.17[P17] variants was higher than that of GII.8[P8] variants (1.22 × 10-3 nucleotide substitutions/site/year to 9.31 × 10-4 nucleotide substitutions/site/year, respectively). The enzyme catalytic reaction demonstrated that the Vmax value of GII.17[P17] RdRp was 2.5 times than that of GII.8[P8] RdRp. And the Km of GII.17[P17] and GII.8[P8] RdRp were 0.01 and 0.15 mmol/L, respectively. Then, GII.8[P8] RdRp fragment mutants (A-F) were designed, among which GII.8[P8]-A/B containing the conserved motif G/F were found to have significant effects on improving RdRp activity. The Km values of GII.8[P8]-A/B reached 0.07 and 0.06 mmol/L, respectively. And their Vmax values were 1.34 times than that of GII.8[P8] RdRp. In summary, our results suggested that RdRp activities were correlated with their epidemic characteristics. These findings will ultimately provide a better understanding in replication mechanism of noroviruses and development of antiviral drugs.

Performance and transcriptome analysis of Salmonella enterica serovar Enteritidis PT 30 under persistent desiccation stress: Cultured by lawn and broth methods.

Lawn-harvest method uses a solid medium (e.g., tryptic soy agar, TSA) to produce bacterial lawns and is widely accepted for the culture of microorganisms in microbial studies of low-moisture foods (LMFs, foods with water activity less than 0.85). It produces desiccation-tolerant cells with higher D-values in LMFs; however, little is known about the molecular mechanisms underlying bacterial resistance. Salmonella enterica Enteritidis PT 30 (S. Enteritidis), the most pertinent pathogen in LMFs, was cultured in TSA and tryptic soy broth (TSB). Cells were harvested and inoculated on filter papers to assess their performance under a relative humidity of 32 ± 2%. Transcriptome analysis of cultured cells during long-term desiccation (24, 72, and 168 h) was conducted in TruSeq PE Cluster Kit (Illumina) by paired-end methods. Lawn-cultured S. Enteritidis cells have stronger survivability (only decreased by 0.78 ± 0.12 log after 130 d of storage) and heat tolerance (higher D/ß value) than those from the broth method. More desiccation genes of lawn-cultured cells were significantly upregulated from growth to long-term desiccation. Differentially expressed genes were the most enriched in the ribosome and sulfur metabolism pathways in the lawn- and broth-cultured groups. This study tracked the transcriptomic differences between two cultured groups in response to long-term desiccation stress and revealed some molecular mechanisms underlying their different suitability in microbial studies of LMFs.

Effect of prone position in patients with acute respiratory distress syndrome supported by venovenous extracorporeal membrane oxygenation: a retrospective cohort study.

BACKGROUND: The application of prone position (PP) in acute respiratory distress syndrome (ARDS) supported by venovenous extracorporeal membrane oxygenation (VV-ECMO) is controversial. OBJECTIVES: To evaluate the safety and efficacy of application of PP during VV-ECMO in patients with ARDS. METHODS: This was a single-center, retrospective study of patients who met the Berlin definition of ARDS, and were supported with VV-ECMO. We divided the patients into two groups. The prone group included patients who were supported by VV-ECMO, and experienced at least one period of PP, while those without PP during VV-ECMO were defined as the supine group. Propensity score matching (PSM) at a ratio of 1:1 was introduced to minimize potential confounders. The primary outcomes were the complications of PP and the change of arterial oxygen pressure/fraction of the inspiration (PaO2/FiO2) ratio after PP. The secondary outcomes were hospital survival, ICU survival, and ECMO weaning rate. RESULTS: From April 2013 to October 2020, a total of 91 patients met the diagnostic criteria of ARDS who were supported with ECMO. 38 patients (41.8%) received at least one period of PP during ECMO, while 53 patients (58.2%) were maintained in supine position during ECMO. 22 minor complications were reported in the prone group and major complications were not found. The other ECMO-related complications were similar between two groups. The PaO2/FiO2 ratio significantly improved after PP compared with before (174.50 (132.40-228.25) mmHg vs. 158.00 (122.93-210.33) mmHg, p < 0.001). PSM selected 25 pairs of patients with similar characteristics. Hospital survival or ICU survival did not differ between the two groups (40% vs. 28%, p = 0.370; 40% vs. 32%, p = 0.556). Significant difference of ECMO weaning rate between two groups was not found (56% vs. 32%, p = 0.087). CONCLUSIONS: PP during VV-ECMO was safe and could improve oxygenation. A large-scale and well-designed RCT is needed in the future.

The population pharmacokinetics and dose optimization of polymyxin B in critically ill patients with or without extracorporeal membrane oxygenation.

WHAT IS KNOWN AND OBJECTIVE: The presence of extracorporeal membrane oxygenation (ECMO) is suggested to further exacerbate the pharmacokinetics (PK) alterations that occur during critical illness. The objectives of this study were to determine the population PK model of polymyxin B in critically ill patients with or without ECMO support investigated the influence of ECMO on PK variability and to identify an optimal dosing strategy. METHODS: Forty-four critically ill patients were enrolled, including eight patients with ECMO support. Eight serial serum samples were collected from each patient at steady state. The population PK was determined using NONMEM and Monte Carlo simulation was performed to evaluate the exposures of different dosing regimens. RESULTS: The PK analyses included 342 steady-state concentrations and a two-compartment model was optimal for polymyxin B PK data modelling. In the final model, creatinine clearance (CLCR ) was the significant covariate on CL (typical value 1.27 L/h; between-subject variability 15.1%) and ECMO did not show a significant impact on the polymyxin B PK. Additionally, we found that the PK parameter estimates of patients with and without ECMO support were mostly similar. Based on Monte Carlo simulations, the dose escalation of polymyxin B in patients with increased CLCR improved the probability of achieving required exposure. For patients with CLCR ≤ 120 ml/min, a dosage regimen of 100 mg every 12 h may represent the optimal regimen at an MIC of 1 mg/L. WHAT IS NEW AND CONCLUSION: The impact of ECMO on the polymyxin B PK is likely to be minimal. Our study showed a potential relationship between CLCR and polymyxin B CL, and the dose of polymyxin B should be adjusted in patients with increased CLCR .

SMRT and Illumina RNA-Seq Identifies Potential Candidate Genes Related to the Double Flower Phenotype and Unveils SsAP2 as a Key Regulator of the Double-Flower Trait in Sagittaria sagittifolia.

Double flowers are one of the important objectives of ornamental plant breeding. Sagittaria sagittifolia is an aquatic herb in the Alismataceae family that is widely used as an ornamental plant in gardens. However, the reference genome has not been published, and the molecular regulatory mechanism of flower formation remains unclear. In this study, single molecule real-time (SMRT) sequencing technology combined with Illumina RNA-Seq was used to perform a more comprehensive analysis of S. sagittifolia for the first time. We obtained high-quality full-length transcripts, including 53,422 complete open reading frames, and identified 5980 transcription factors that belonged to 67 families, with many MADS-box genes involved in flower formation being obtained. The transcription factors regulated by plant hormone signals played an important role in the development of double flowers. We also identified an AP2 orthologous gene, SsAP2, with a deletion of the binding site for miR172, that overexpressed SsAP2 in S. sagittifolia and exhibited a delayed flowering time and an increased number of petals. This study is the first report of a full-length transcriptome of S. sagittifolia. These reference transcripts will be valuable resources for the analysis of gene structures and sequences, which provide a theoretical basis for the molecular regulatory mechanism governing the formation of double flowers.

An Investigation on the Occurrence and Molecular Characterization of Bacillus cereus in Meat and Meat Products in China.

Bacillus cereus is a common foodborne pathogen that can cause both gastrointestinal and nongastrointestinal diseases. In this study, we collected 603 meat and meat products from 39 major cities in China. The positive contamination rate of B. cereus in the collected samples was 26.37% (159/603), and the contamination level in 5.03% (8/159) positive samples exceeded 1100 most probable number/g. The detection rates of virulence genes were 89.7% for the nheABC gene group, 37.1% for the hblACD gene cluster, 82.3% for cytK-2, and 2.9% for cesB. Notably, all isolates presented with multiple antibiotic resistance, and 99.43% of isolates were resistant to five classes of antibiotics. In addition, the multilocus sequence typing results indicated that all isolates were rich in genetic diversity. Collectively, we conducted a systematic investigation on the prevalence and characterization of B. cereus in meat and meat products in China, providing crucial information for assessing the risk of B. cereus occurrence in meat and meat products.

Identification and Characterization of Glucosyltransferase That Forms 1-Galloyl-ß-d-Glucogallin in Canarium album L., a Functional Fruit Rich in Hydrolysable Tannins.

Hydrolysable tannins (HTs) are useful secondary metabolites that are responsible for pharmacological activities and astringent taste, flavor, and quality in fruits. They are also the main polyphenols in Canarium album L. (Chinese olive) fruit, an interesting and functional fruit that has been cultivated for over 2000 years. The HT content of C. album fruit was 2.3-13 times higher than that of berries with a higher content of HT. 1-galloyl-ß-d-glucose (ßG) is the first intermediate and the key metabolite in the HT biosynthesis pathway. It is catalyzed by UDP-glucosyltransferases (UGTs), which are responsible for the glycosylation of gallic acid (GA) to form ßG. Here, we first reported 140 UGTs in C. album. Phylogenetic analysis clustered them into 14 phylogenetic groups (A, B, D-M, P, and Q), which are different from the 14 typical major groups (A~N) of Arabidopsis thaliana. Expression pattern and correlation analysis showed that UGT84A77 (Isoform0117852) was highly expressed and had a positive correlation with GA and ßG content. Prokaryotic expression showed that UGT84A77 could catalyze GA to form ßG. These results provide a theoretical basis on UGTs in C. album, which will be helpful for further functional research and availability on HTs and polyphenols.

Genetic characteristics and virulence of Listeria monocytogenes isolated from fresh vegetables in China.

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.

Prevalence and genetic diversity of human sapovirus associated with sporadic acute gastroenteritis in South China from 2013 to 2017.

Human sapovirus (SaV) is an important viral agent for acute diarrhea worldwide, but timely prevalence data of human SaV in South China are still lacking. In this study, a 4-year surveillance was conducted to characterize the prevalence and genetic characteristics of the circulating SaV associated with sporadic diarrhea in South China. From November 2013 to October 2017, 569 fecal samples from patients with acute diarrhea were collected. SaV was detected in 11 samples with a positive rate of 1.93%. Three human genogroups of GI, GII, and GIV were identified, including five GI.1 strains, three GI.2 strains, one GI.3 strain, one GII.8 strain, and one GIV strain. Furthermore, multiple alignments of complete capsid protein VP1 genes of five local GI.1 strains and other available GI.1 strains in GenBank were performed. Average pairwise identities were calculated at 95.33% and 99.36% at nucleotide and amino acid levels, and only six variable amino acid sites were found during its 36-years' evolution process. GI.1 strains could be further phylogenetically divided into four clusters with an approximate temporal evolution pattern, and local strains belonged to Cluster-d with other four strains from China and Japan. In summary, SaV was identified as an etiological agent responsible for sporadic gastroenteritis in Guangzhou with a low prevalence rate as in other Chinese cities, but its high genetic diversity suggested the necessity of continuous SaV surveillance in the future.

Isolation and Structure of Cancer Cell Growth Inhibitory Tetracyclic Triterpenoids from the Zimbabwean Monadenium lugardae.

The Zimbabwean medicinal plant Monadenium lugardae was evaluated as a potential source of new anticancer constituents. Four new tetracyclic triterpene (1-4) were isolated, accompanied by four previously known triterpenes (5-8). Against a panel of human tumor cell lines, lugardstatins 1 (1) and 2 (2) had good cancer cell growth inhibitory activity. All of the triterpene structures (1-8) were established by 1D and 2D NMR spectrometric and HR mass spectrometric analysis.

Identification of MYB Transcription Factor, a Regulator Related to Hydrolysable Tannin Synthesis in Canarium album L., and Functional Analysis of CaMYBR04.

Hydrolysed tannins (HTs) are polyphenols, which are related to the astringency, flavour, colour, stability, medicinal value and other characteristics of many fruits and vegetables. The biosynthetic mechanism of the majority of HTs remains unknown, and many biosynthetic pathways of HTs are speculative conclusions that have not been confirmed. The fruit of Canarium album L. (Chinese olive), which is notable for its pharmacological and edible properties, is rich in HTs. The fruit has a distinctive bitter and astringent taste when initially consumed, which mellows to a sweet sensation upon chewing. HTs serve as the primary material basis for the formation of the Chinese olive fruit's astringent quality and pharmacological effects. In this study, the fruit of C. album Changying was utilised as the research material. The objective of this study was to provide a theoretical basis for the quality control of Chinese olive fruit and the application and development of its medicinal value. In addition, the study aimed to identify and screen related MYB transcription factors involved in the synthesis of HTs in the fruit and to clarify the mechanism of MYBs in the process of synthesis and regulation of HTs in Chinese olive fruit. The principal findings were as follows. A total of 83 differentially expressed Chinese olive MYB transcription factors (CaMYBs) were identified, including 54 1R-MYBs (MYB-related), 25 2R-MYBs (R2R3-MYBs), 3 3R-MYBs, and 1 4R-MYB. Through trend analysis and correlation analysis, it was found that CaMYBR04 (Isoform0032534) exhibited a significantly higher expression (FPKM) than the other CaMYBs. The full-length cDNA sequence of CaMYBR04 was cloned and transformed into strawberry. The results demonstrated that CaMYBR04 significantly enhanced the fruit's hydrolysable tannin content. Consequently, this study elucidated the function of CaMYBR04, a regulator of the Chinese olive fruit hydrolysable tannin synthesis pathway, and established a theoretical foundation for the synthesis and regulation of fruit HTs.

Renal Replacement Therapy as a New Indicator of Voriconazole Clearance in a Population Pharmacokinetic Analysis of Critically Ill Patients.

AIMS: The pharmacokinetic (PK) profiles of voriconazole in intensive care unit (ICU) patients differ from that in other patients. We aimed to develop a population pharmacokinetic (PopPK) model to evaluate the effects of using extracorporeal membrane oxygenation (ECMO) and continuous renal replacement therapy (CRRT) and those of various biological covariates on the voriconazole PK profile. METHODS: Modeling analyses of the PK parameters were conducted using the nonlinear mixed-effects modeling method (NONMEM) with a two-compartment model. Monte Carlo simulations (MCSs) were performed to observe the probability of target attainment (PTA) when receiving CRRT or not under different dosage regimens, different stratifications of quick C-reactive protein (qCRP), and different minimum inhibitory concentration (MIC) ranges. RESULTS: A total of 408 critically ill patients with 746 voriconazole concentration-time data points were included in this study. A two-compartment population PK model with qCRP, CRRT, creatinine clearance rate (CLCR), platelets (PLT), and prothrombin time (PT) as fixed effects was developed using the NONMEM. CONCLUSIONS: We found that qCRP, CRRT, CLCR, PLT, and PT affected the voriconazole clearance. The most commonly used clinical regimen of 200 mg q12h was sufficient for the most common sensitive pathogens (MIC ≤ 0.25 mg/L), regardless of whether CRRT was performed and the level of qCRP. When the MIC was 0.5 mg/L, 200 mg q12h was insufficient only when the qCRP was <40 mg/L and CRRT was performed. When the MIC was ≥2 mg/L, a dose of 300 mg q12h could not achieve ≥ 90% PTA, necessitating the evaluation of a higher dose.

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

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

Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances.

The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

A systematic review and meta-analysis indicates a high risk of human noroviruses contamination in vegetable worldwide, with GI being the predominant genogroup.

Human noroviruses (HuNoVs) are the most predominant viral agents of acute gastroenteritis. Vegetables are important vehicles of HuNoVs transmission. This study aimed to assess the HuNoVs prevalence in vegetables. We searched the Web of Science, Excerpta Medica Database, PubMed, and Cochrane databases until June 1, 2023. A total of 27 studies were included for the meta-analysis. Statistical analysis was conducted using Stata 14.0 software. This analysis showed that the pooled HuNoVs prevalence in vegetables was 7 % (95 % confidence interval (CI): 3-13) worldwide. The continent with largest number of studies was Europe, and the highest number of samples was lettuce. As revealed by the results of the subgroup meta-analysis, the prevalence of GI genogroup was the highest (3 %, 95 % CI: 1-7). A higher prevalence was seen in vegetables from farms (18 %, 95 % CI: 5-37), while only 4 % (95 % CI: 1-8) in retail. The HuNoVs prevalence of ready-to-eat vegetables and non-ready-to-eat vegetables was 2 % (95 % CI: 0-8) and 9 % (95 % CI: 3-16), respectively. The prevalence by quantitative real time RT-PCR was 8 % (95 % CI: 3-15) compared to 3 % (95 % CI: 0-13) by conventional RT-PCR. Furthermore, the HuNoVs prevalence in vegetables was 6 % (95 % CI: 1-14) in ISO pretreatment method and 8 % (95 % CI: 1-19) in non-ISO method, respectively. This study is helpful in comprehensively understanding the prevalence of HuNoVs contamination in vegetables worldwide.

Enhanced heat tolerance of freeze-dried Enterococcus faecium NRRL B-2354 as valid Salmonella surrogate in low-moisture foods.

In microbial studies of low-moisture foods (LMFs, water activity less than 0.85), freeze-dried bacteria benefit us to inoculate LMFs without introducing extra water or altering food physiochemical properties. However, the freeze-drying process would bring unavoidable damage to bacterial cells and results in less-resistant inoculum that are unlikely to be qualified in microbial studies. Herein, we enhanced bacterial heat tolerance by subjecting the cells to mild heat (42-50 °C) to counteract the reduced heat tolerance and survivability of freeze-dried bacteria. Enterococcus faecium NRRL B-2354 (E. faecium), a Salmonella surrogate in LMFs, was used as the target microorganism because it was widely accepted in microbial validation of thermal pasteurizing LMFs. Three types of LMFs (peanut powder, protein powder, and onion powder) were used as LMFs models to validate the freeze-dried E. faecium in comparison with Salmonella enterica Enteritidis PT 30 (S. Enteritidis) prepared by the traditional aqueous method. The heat tolerance (D65℃ value) of E. faecium increased at all treatments and peaked (+31.48 ± 0.13%) at temperature-time combinations of 45 °C-60 min and 50 °C-5 min. Survivability of freeze-dried inoculum and its heat tolerance retained well within 50 d storage. The freeze-dried E. faecium was prepared in this study brought equal or higher heat tolerance (D85℃ or D75℃) than S. Enteritidis in tested LMFs models. For instance, the D85℃ of freeze-dried E. faecium (heat-treated at 50 °C for 5 min) and S. Enteritidis in whole egg powder are 35.56 ± 1.52 min and 28.41 ± 0.41 min, respectively. The freeze-dried E. faecium with enhanced heat tolerance appears to be a suitable Salmonella surrogate for dry-inoculating LMFs. Our protocol also enables industry-scale production of freeze-dried inoculum by broth-cultivation method combined with mild-heat treatment.

Occurrence, molecular characterization and antibiotic resistance of Cronobacter spp. isolated from wet rice and flour products in Guangdong, China.

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.