Prevalence, Antimicrobial Resistance, and Whole Genome Sequencing Analysis of Shiga Toxin-Producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) from Imported Foods in China during 2015-2021.

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

Toll-like receptor 4-induced ryanodine receptor 2 oxidation and sarcoplasmic reticulum Ca2+ leakage promote cardiac contractile dysfunction in sepsis.

Studies suggest the potential role of a sarcoplasmic reticulum (SR) Ca2+ leak in cardiac contractile dysfunction in sepsis. However, direct supporting evidence is lacking, and the mechanisms underlying this SR leak are poorly understood. Here, we investigated the changes in cardiac Ca2+ handling and contraction in LPS-treated rat cardiomyocytes and a mouse model of polymicrobial sepsis produced by cecal ligation and puncture (CLP). LPS decreased the systolic Ca2+ transient and myocyte contraction as well as SR Ca2+ content. Meanwhile, LPS increased Ca2+ spark-mediated SR Ca2+ leak. Preventing the SR leak with ryanodine receptor (RyR) blocker tetracaine restored SR load and increased myocyte contraction. Similar alterations in Ca2+ handling were observed in cardiomyocytes from CLP mice. Treatment with JTV-519, an anti-SR leak drug, restored Ca2+ handling and improved cardiac function. In the LPS-treated cardiomyocytes, mitochondrial reactive oxygen species and oxidative stress in RyR2 were increased, whereas the levels of the RyR2-associated FK506-binding protein 1B (FKBP12.6) were decreased. The Toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 reduced the oxidative stress in LPS-treated cells, decreased the SR leak, and normalized Ca2+ handling and myocyte contraction. Consistently, TLR4 deletion significantly improved cardiac function and corrected abnormal Ca2+ handling in the CLP mice. This study provides evidence for the critical role of the SR Ca2+ leak in the development of septic cardiomyopathy and highlights the therapeutic potential of JTV-519 by preventing SR leak. Furthermore, it reveals that TLR4 activation-induced mitochondrial reactive oxygen species production and the resulting oxidative stress in RyR2 contribute to the SR Ca2+ leak.

Resveratrol and polydatin as modulators of Ca2+ mobilization in the cardiovascular system.

In the cardiovascular system, Ca2+ controls cardiac excitation-contraction coupling and vascular contraction and dilation. Disturbances in intracellular Ca2+ homeostasis induce malfunctions of the cardiovascular system, including cardiac pump dysfunction, arrhythmia, remodeling, and apoptosis, as well as hypertension and impairment of vascular reactivity. Therefore, developing drugs and strategies manipulating Ca2+ handling are highly valued in the treatment of cardiovascular disease. Resveratrol (Res) and polydatin (PD), a Res glucoside, have been well established to have beneficial effects on improving cardiovascular function. Studies from our laboratory and others have demonstrated that they exhibit inotropic effects on normal heart and therapeutic effects on hypertension, cardiac ischemia/reperfusion injury, hypertrophy, and heart failure by manipulating Ca2+ mobilization. The actions of Res and PD on Ca2+ signals delicately manipulated by multiple Ca2+ -handling proteins are pleiotropic and somewhat controversial, depending on cellular species and intracellular oxidative status. Here, we focus on the effects of Res and PD on controlling Ca2+ homeostasis in the heart and vasculature under normal and diseased conditions and highlight the key direct and indirect molecules mediating these effects.

Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

Objectives: To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . Methods: The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Results: Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. Conclusions: The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

Ultrasensitive detection and rapid identification of multiple foodborne pathogens with the naked eyes.

In this study, a novel approach for ultrasensitive detection and rapid high-throughput identification of a panel of common foodborne pathogens with the naked eyes is presented. As a proof-of-concept application, a multiple pathogen analysis array is fabricated through immobilizing three specific polyT-capture probes which can respectively recognize rfbE gene (Escherichia coli O157:H7), invA gene (Salmonella enterica), inlA gene (Listeria monocytogenes) on the plastic substrates. PCR has been developed for amplification and labeling target genes of rfbE, invA, inlA with biotin. The biotinated target DNA is then captured onto the surface of plastic strips through specific DNA hybridization. The succeeding staining of biotinated DNA duplexes with avidin-horseradish peroxidise (AV-HRP) and biotinated anti-HRP antibody greatly amplifies the detectable signal through the multiple cycle signal amplification strategy, and thus realizing ultrasensitive and specific detection of the above three pathogens in food samples with the naked eyes. Results showed approximately 5 copies target pathogenic DNA could be detected with the naked eyes. This simple but very efficient colorimetric assay also show excellent anti-interference capability and good stability, and can be readily applied to point-of-care diagnosis.

A "three-in-one" sample preparation method for simultaneous determination of B-group water-soluble vitamins in infant formula using VitaFast(®) kits.

VitaFast(®) test kits designed for the microbiological assay in microtiter plate format can be applied to quantitative determination of B-group water-soluble vitamins such as vitamin B12, folic acid and biotin, et al. Compared to traditional microbiological methods, VitaFast(®) kits significantly reduce sample processing time and provide greater reliability, higher productivity and better accuracy. Recently, simultaneous determination of vitamin B12, folic acid and biotin in one sample is urgently required when evaluating the quality of infant formulae in our practical work. However, the present sample preparation protocols which are developed for individual test systems, are incompatible with simultaneous determination of several analytes. To solve this problem, a novel "three-in-one" sample preparation method is herein developed for simultaneous determination of B-group water-soluble vitamins using VitaFast(®) kits. The performance of this novel "three-in-one" sample preparation method was systematically evaluated through comparing with individual sample preparation protocols. The experimental results of the assays which employed "three-in-one" sample preparation method were in good agreement with those obtained from conventional VitaFast(®) extraction methods, indicating that the proposed "three-in-one" sample preparation method is applicable to the present three VitaFast(®) vitamin test systems, thus offering a promising alternative for the three independent sample preparation methods. The proposed new sample preparation method will significantly improve the efficiency of infant formulae inspection.

Detection of single-digit foodborne pathogens with the naked eye using carbon nanotube-based multiple cycle signal amplification.

A carbon nanotube (CNT)-based multiple cycle signal amplification strategy has been demonstrated for detection of single-digit foodborne pathogens with the naked eye. In the present design, CNTs are used as carriers for loading numerous horseradish peroxidase (HRP) and concanavalin A (ConA) tags, and multiple cycle signal amplification is achieved through the biotinylated anti-HRP antibody and avidin-HRP.

Polydatin (PD) inhibits IgE-mediated passive cutaneous anaphylaxis in mice by stabilizing mast cells through modulating Ca²âº mobilization.

Mast cells play a key role in the pathogenesis of asthma and are a promising target for therapeutic intervention in asthma. This study investigated the effects of polydatin (PD), a resveratrol glucoside, on mast cell degranulation upon cross-linking of the high-affinity IgE receptors (FcεRI), as well as the anti-allergic activity of PD in vivo. Herein, we demonstrated that PD treatment for 30 min suppressed FcεRI-mediated mast cell degranulation in a dose-dependent manner. Concomitantly, PD significantly decreased FcεRI-mediated Ca²âº increase in mast cells. The suppressive effects of PD on FcεRI-mediated Ca²âº increase were largely inhibited by using LaCl3 to block the Ca²âº release-activated Ca²âº channels (CRACs). Furthermore, PD significantly inhibited Ca²âº entry through CRACs evoked by thapsigargin (TG). Knocking down protein expression of Orai1, the pore-forming subunit of CRACs, significantly decreased PD suppression of FcεRI-induced intracellular Ca²âº influx and mast cell degranulation. In a mouse model of mast cell-dependent passive cutaneous anaphylaxis (PCA), in vivo PD administration suppressed mast cell degranulation and inhibited anaphylaxis. Taken together, our data indicate that PD stabilizes mast cells by suppressing FcεRI-induced Ca²âº mobilization mainly through inhibiting Ca²âº entry via CRACs, thus exerting a protective effect against PCA.

Lipopolysaccharide enhances FcεRI-mediated mast cell degranulation by increasing Ca2+ entry through store-operated Ca2+ channels: implications for lipopolysaccharide exacerbating allergic asthma.

Lipopolysaccharide (LPS) can exacerbate asthma; however, the mechanisms are not fully understood. This study investigated the effect of LPS on antigen-stimulated mast cell degranulation and the underlying mechanisms. We found that LPS enhanced degranulation in RBL-2H3 cells and mouse peritoneal mast cells upon FcεRI activation, in a dose- and time-dependent manner. Parallel to the alteration of degranulation, LPS increased FcεRI-activated Ca(2+) mobilization, as well as Ca(2+) entry through store-operated calcium channels (SOCs) evoked by thapsigargin. Blocking Ca(2+) entry through SOCs completely abolished LPS enhancement of mast cell degranulation. Consistent with functional alteration of SOCs, LPS increased mRNA and protein levels of Orai1 and STIM1, two major subunits of SOCs, in a time-dependent manner. In addition, LPS increased the mRNA level of Toll-like receptor 4 (TLR4) in a time-dependent manner. Blocking TLR4 with Cli-095 inhibited LPS, increasing transcription and expression of SOC subunits. Concomitantly, the effect of LPS enhancement of Ca(2+) mobilization and mast cell degranulation was largely reduced by Cli-095. Administration of LPS (1 µg) in vivo aggravated airway hyperreactivity and inflammatory reactions in allergic asthmatic mice. Histamine levels in serum and bronchoalveolar lavage fluid were increased by LPS treatment. In addition, Ca(2+) mobilization was enhanced in peritoneal mast cells isolated from LPS-treated asthmatic mice. Taken together, these results imply that LPS enhances mast cell degranulation, which potentially contributes to LPS exacerbating allergic asthma. Lipopolysaccharide increases Ca(2+) entry through SOCs by upregulating transcription and expression of SOC subunits, mainly through interacting with TLR4 in mast cells, resulting in enhancement of mast cell degranulation upon antigen stimulation.