Diverse Genotypes of Cronobacter spp. Associated with Dairy Farm Systems in Jiangsu and Shandong Provinces in China.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

Genomic epidemiology and characterization of Staphylococcus aureus isolates from raw milk in Jiangsu, China: emerging broader host tropism strain clones ST59 and ST398.

Staphylococcus aureus is highly pathogenic and can cause disease in both humans and domestic animals. The aim of this study was to investigate the genomic epidemiology of S. aureus isolates from raw milk in Jiangsu Province, China, to identify predominant lineages and their associated genomic and phenotypic characteristics. In this study, we identified 117 S. aureus isolates collected from 1,062 samples in Jiangsu Province between 2021 and 2022. Based on whole-genome sequencing (WGS) data from 117 S. aureus isolates, molecular analyses indicated CC1-ST1 (26.50%, 31/117), CC97-ST97 (18.80%, 22/117), CC398-ST398 (10.26%, 12/117), CC8-ST630 (7.69%, 9/117) and CC59-ST59 (2.56%, 3/117) were the major lineages. The prevalence of mecA-positive strains was 11.11%. Four methicillin-resistant S. aureus (MRSA) lineages were found, including MRSA-ST59-t172 (n = 3), OS-MRSA-ST398-t011 (n = 1), MRSA-ST630-t2196 (n = 2) and OS-MRSA-ST630-t2196 (n = 7). Phenotypic resistance to penicillin (30.77%, 36/117), ciprofloxacin (17.09%, 20/117) and erythromycin (15.38%, 18/117) was observed which corresponded with resistance genotypes. All of the isolates could produce biofilms, and 38.46% (45/117) of isolates had invasion rates in mammary epithelial cells (MAC-T) of greater than 1%. Interestingly, most biofilm-producing and invading isolates harbored ebp-icaA-icaB-icaC-icaR-clfA-clfB-fnbA-fnbB-sdrC-sdrD-sdrE-map-can (27.35%, 32/117) and ebp-icaA-icaB-icaC-icaD-icaR-clfA-clfB-fnbA-fnbB-sdrC-sdrD-sdrE-map (33.33%, 39/117) adherence-associated gene patterns and belonged to lineages CC1 and CC97, respectively. Virulence factor assays showed that 47.01% of the isolates contained at least enterotoxin genes. Isolates harboring the immune evasion cluster (IEC) genes (sea, sak, chp, and scn) were predominantly categorized as STs 464, 398, and 59. IEC-positive ST398 and ST59 isolates contained a very high proportion of virulence genes located on prophages, whereas most IEC-negative ST398 clade isolates carried broad-spectrum drug resistance genes. Meanwhile, the IEC-positive ST398 clade showed a close genetic relationship with isolates from the pork supply chain and hospital surgical site infections. MRSA-ST59 strains showed the closest genetic relationship with an isolate from quick-frozen products. High-risk livestock-associated strains ST398 and MRSA-ST59 were detected in raw milk, indicating a potential public health risk of S. aureus transmission between livestock and humans. Our study highlights the necessity for S. aureus surveillance in the dairy industry.

Solid-state fermentation with Rhizopus oligosporus RT-3 enhanced the nutritional properties of soybeans.

Fermented soybean products are favorite foods worldwide because of their nutritional value and health effects. In this study, solid-state fermentation (SSF) of soybeans with Rhizopus oligosporus RT-3 was performed to investigate its nutraceutical potential. A rich enzyme system was released during SSF. Proteins were effectively transformed into small peptides and amino acids. The small peptide content increased by 13.64 times after SSF for 60 h. The antioxidant activity of soybeans was enhanced due to the release of phenolic compounds. The soluble phenolic content increased from 2.55 to 9.28 gallic acid equivalent (GAE) mg/g after SSF for 60 h and exhibited high correlations with microbial enzyme activities during SSF. The potential metabolic pathways being triggered during SSF indicated that the improved nutritional composition of soybean attributed to the biochemical reactions catalyzed by microbial enzymes. These findings demonstrated that SSF could evidently improve the nutritional value and prebiotic potential of soybeans.

Antiviral effect of a bacteriophage on murine norovirus replication via modulation of the innate immune response.

Bacteriophages (phages) are viruses of bacteria. Despite the growing progress in research on phage interactions with eukaryotic cells, our understanding of the roles of phages and their potential implications remains incomplete. The objective of this study was to investigate the effects of the Staphylococcus aureus phage vB_SauM_JS25 on murine norovirus (MNV) replication. Experiments were performed using the RAW 264.7 cell line. After phage treatment, MNV multiplication was significantly inhibited, as indicated by real-time quantitative polymerase chain reaction (RT-qPCR) analysis, western blotting, the 50% tissue culture infectious dose and immunofluorescence. Furthermore, we revealed transcriptional changes in phage/MNV co-incubated RAW 264.7 cells through RNA sequencing (RNA-seq) and bioinformatic analysis. Our subsequent analyses revealed that the innate immune response might play an important role in restriction of MNV replication, such as the cellular response to IFN-γ and response to IFN-γ. Additionally, gene expression of IL-10, Arg-1, Ccl22, GBP2, GBP3, GBP5, and GBP7 was increased significantly, which indicated a strong correlation between RT-qPCR and RNA-seq results. Furthermore, phage treatment activated guanylate binding proteins (GBPs), as revealed by RT-qPCR analysis, western blotting, and confocal microscopy. Taken together, these data suggest that the phage affects the innate response, such as the IFN-inducible GTPases and GBPs, and therefore exerts an antiviral effect in vitro. Collectively, our findings provide insights into the interactions of immune cells and phages, which establish phage-based antiviral effects.

Biodiversity of New Lytic Bacteriophages Infecting Shigella spp. in Freshwater Environment.

Bacteriophages, viruses that infect and replicate within prokaryotic cells are the most abundant life forms in the environment, yet the vast majority of them have not been properly reported or even discovered. Almost all reported bacteriophages infecting the Enterobacteriaceae family, with Escherichia coli being the major subject of studies, have been isolated from wastewater, sewage, and effluent resources. In the present study, we focused on the distribution and biodiversity of Shigella phages in an aquatic ecosystem. While no Shigella bacteria was recovered from the Yangtze River, three lytic phages were isolated from this ecosystem and were subjected to biological, morphological, and genomic characteristics. Comparative genomics and phylogenetic analyses demonstrated that vB _SflM_004 isolate belongs to Myoviridae family, Felixounavirus genus of Ounavirinae subfamily, vB_SdyM_006 was classified under the same family, however, it is suggested to be in a new genus under Tevenvirinae subfamily with some other related bacteriophages. vB_SsoS_008 phage belongs to the Siphoviridae family, Tunavirus genus, Tunavirinae subfamily. The phages did not harbor any genes involved in the lysogenic cycles and showed a high temperature and pH stability. The biodiversity of the isolated phages highly suggests that continued isolation on non-model members of Enterobacteriaceae family is necessary to fully understand bacteriophage diversity in aquatic environments.

Stabilization of Liposomes by Perfluorinated Compounds.

Perfluorinated compounds (PFCs) are emerging persistent environmental contaminants that may be toxic to animals and humans. To gain fundamental insights into the mechanism of their toxicity, the interactions of phosphocholine (PC) liposomes as model membranes were studied with three types of PFCs, including perfluorooctanoic acid, perfluorooctane sulfonate, and perfluorohexanesulfonic acid potassium salt, together with three common surfactants: sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and sodium 1-heptanesulfonate (SHS). The interactions were systematically characterized by zeta potential measurement, dynamic light scattering, negative-stain transmission electron microscopy, and fluorescence spectroscopy. Unmodified liposomes, calcein-loaded liposomes, and Laurdan dye-embedded liposomes were all tested. By gradually increasing the temperature, the three PFCs and SHS decreased the leakage of calcein-loaded 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes, whereas SDS and CTAB increased the leakage. The PFCs that affected the lipid membranes stronger than SHS were attributable to their perfluoroalkyl carbon chains. Packing of the lipids was further studied using Laurdan dye as a probe. Calcein leakage tests also indicated that PFCs inhibited lipid membrane leakage induced by inorganic nanoparticles such as silica and gold nanoparticles. This study confirmed the similar effect of the PFCs as cholesterol in affecting membrane properties and would be helpful for understanding the interaction mechanism of PFCs and cell membranes.

Paper-based immune-affinity arrays for detection of multiple mycotoxins in cereals.

Mycotoxins produced by different species of fungi may coexist in cereals and feedstuffs, and could be highly toxic for humans and animals. For quantification of multiple mycotoxins in cereals, we developed a paper-based mycotoxin immune-affinity array. First, paper-based microzone arrays were fabricated by photolithography. Then, monoclonal mycotoxin antibodies were added in a copolymerization reaction with a cross-linker to form an immune-affinity monolith on the paper-based microzone array. With use of a competitive immune-response format, paper-based mycotoxin immune-affinity arrays were successfully applied to detect mycotoxins in samples. The detection limits for deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin were 62.7, 10.8, 0.36, and 0.23 µg·kg-1, respectively, which meet relevant requirements for these compounds in food. The recovery rates were 81-86% for deoxynivalenol, 89-117% for zearalenone, 79-86% for T-2 toxin, and 78-83% for HT-2 toxin, and showed the paper-based immune-affinity arrays had good reproducibility. In summary, the paper-based mycotoxin immune-affinity array provides a sensitive, rapid, accurate, stable, and convenient platform for detection of multiple mycotoxins in agro-foods. Graphical abstract Paper-based immune-affinity monolithic array. DON deoxynivalenol, HT-2 HT-2 toxin, T-2 T-2 toxin, PEGDA polyethylene glycol diacrylate, ZEN zearalenone.

Development of immune-affinity 96 spots monolith array for multiple mycotoxins detection in food samples.

In this paper, a novel highly sensitive chemiluminescence immune-affinity 96 spots monolith array was developed to detect deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), and fumonisin B1 (FB1) in corn samples. Firstly, the monolith array was prepared through on suit UV-initiated copolymerization using polyethylene glycol diacrylate (PEGDA) as cross-linker, glycidyl methacrylate (GMA) as functional monomer and polyethylene glycol 200 (PEG 200) as the porogen. Subsequently, the four mycotoxins immune-affinity monolith array was prepared by immobilization of DON, ZEN, T-2, and FB1 antibody. The mole ratio of PEGDA/GMA, UV exposure time, and the volume ratio of PEG 200/PEGDA were optimized to improve the performances of the immune-affinity monolith array. For the mycotoxins immune-affinity monolith array based on chemiluminescence detection, the limit of detection was 0.0036ng/mL (DON), 0.0048ng/mL (ZEN), 0.0039ng/mL (T-2), and 0.0017ng/mL (FB1), respectively. The linear response in the range of 0.01-0.1ng/mL (R(2)=0.98). The results showed that the proposed four mycotoxins immune-affinity monolith array was a stable, accurate, and highly sensitive method to determine levels of DON, ZEN, T-2, and FB1 in real samples.

Separation and analysis of cis-diol-containing compounds by boronate affinity-assisted micellar electrokinetic chromatography.

Cis-diol-containing compounds (CDCCs) are usually highly hydrophilic compounds and are therefore difficult to separate by conventional reversed-phase-based micellar electrokinetic chromatography (MEKC) due to poor selectivity. Here, we report a new method, called boronate affinity-assisted micellar electrokinetic chromatography (BAA-MEKC), to solve this issue. A boronic acid with a hydrophobic alkyl chain was added to the background electrolyte, which acted as a modifier to adjust the selectivity. CDCCs can covalently react with the boronic acid to form negatively charged surfactant-like complexes, which can partition into micelles formed with a cationic surfactant. Thus, CDCCs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase. To verify this method, eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCCs. The effects of experimental conditions on the separation were investigated. Finally, the BAA-MEKC method was applied to the separation and analysis of nucleosides extracted from human urine. BAA-MEKC exhibited better selectivity and improved separation as compared with conventional MEKC and CZE. Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.

Probing the interactions between boronic acids and cis-diol-containing biomolecules by affinity capillary electrophoresis.

The affinity of boronic acids to cis-diol-containing biomolecules has found wide applications in many fields, such as sensing, separation, drug delivery, and functional materials. A sound understanding of the binding interactions will greatly facilitate exquisite applications of this chemistry. Although a few analytical tools have been available for the characterization of the interactions, these techniques are associated with some apparent drawbacks, so they are only applicable to a limited range of boronic acids and cis-diol-containing biomolecules. Therefore, a widely applicable method is still greatly needed. In this work, an affinity capillary electrophoresis (ACE) method was established and validated to probe the interactions between boronic acids and cis-diol-containing biomolecules. The method was proven to be applicable to almost all types of cis-diol-containing biomolecules and boronic acids. Based on this method, a quantitative, comparative study on the interactions between 14 boronic acids that have important potentials for application with 5 typical monosaccharides of biological importance was carried out. The findings provided new insights into boronate affinity interactions, particularly the relationship between the binding strength with the molecular structures of the binding species. Besides, effects of pH and temperature on the binding strength were also investigated. This method exhibited several significant advantages, including (1) possibility of simultaneous study of multiple interactions, (2) low requirement on the purity of the binding species, (3) wide applicability, and (4) high accuracy and precision.

Preparation of organic-silica hybrid boronate affinity monolithic column for the specific capture and separation of cis-diol containing compounds.

A new boronate-silica hybrid monolithic column was prepared using a one-pot approach with 3-acrylamidophenylboronic acid (AAPBA) as the boronate affinity ligand. The AAPBA-silica monolith exhibited several attractive advantages. First, it is highly hydrophilic, providing excellent specificity and avoiding the presence of organic solvent in the mobile phase. Second, due to its large surface area, it exhibited a high binding capacity, 49.5 µmol/mL, the highest among the boronate affinity monolithic columns appeared in the literature. Third, the monolith can bind with cis-diol containing compounds at pH as low as 6.5, which not only avoids the use of basic pH conditions at which the silica monolith may hydrolysis but also facilitates the applications to wider sample range. Finally, the hybrid monolithic column exhibited apparent secondary separation capability, which allows for two-dimensional (2D) separation of cis-diol compounds in a single column. Due to these merits, the AAPBA-silica hybrid monolithic column can be a promising separation medium for the analysis of cis-diol containing compounds.

A benzoboroxole-functionalized monolithic column for the selective enrichment and separation of cis-diol containing biomolecules.

A benzoboroxole-functionalized monolithic column was synthesized, which exhibited the best specificity and affinity towards cis-diol containing biomolecules as compared with the boronate affinity monolithic columns reported as well as significant secondary separation capability under acidic conditions.

Immuno-magnetic beads-based extraction-capillary zone electrophoresis-deep UV laser-induced fluorescence analysis of erythropoietin.

Erythropoietin (EPO) is an important glycoprotein hormone. Recombinant human EPO (rhEPO) is an important therapeutic drug and can be also used as doping reagent in sports. The analysis of EPO glycoforms in pharmaceutical and sports areas greatly challenges analytical scientists from several aspects, among which sensitive detection and effective and facile sample preparation are two essential issues. Herein, we investigated new possibilities for these two aspects. Deep UV laser-induced fluorescence detection (deep UV-LIF) was established to detect the intrinsic fluorescence of EPO while an immuno-magnetic beads-based extraction (IMBE) was developed to specifically extract EPO glycoforms. Combined with capillary zone electrophoresis (CZE), CZE-deep UV-LIF allows high resolution glycoform profiling with improved sensitivity. The detection sensitivity was improved by one order of magnitude as compared with UV absorbance detection. An additional advantage is that the original glycoform distribution can be completely preserved because no fluorescent labeling is needed. By combining IMBE with CZE-deep UV-LIF, the overall detection sensitivity was 1.5 × 10⁻8 mol/L, which was enhanced by two orders of magnitude relative to conventional CZE with UV absorbance detection. It is applicable to the analysis of pharmaceutical preparations of EPO, but the sensitivity is insufficient for the anti-doping analysis of EPO in blood and urine. IMBE can be straightforward and effective approach for sample preparation. However, antibodies with high specificity were the key for application to urine samples because some urinary proteins can severely interfere the immuno-extraction.