Integrated aquaculture contributes to the transfer of mcr-1 between animals and humans via the aquaculture supply chain

Background Since its discovery in 2015, the mobile colistin resistance gene mcr-1 has been reported in bacteria from > 50 countries. Although aquaculture-associated bacteria may act as a significant reservoir for colistin resistance, systematic investigations of mcr-1 in the aquaculture supply chain are scarce. Objectives We investigated the presence of colistin resistance determinants in the aquaculture supply chain in south China and determined their characteristics and relationships. Methods A total of 250 samples were collected from a duck-fish integrated fishery, slaughter house, and market in Guangdong Province, China, in July 2017. Colistin-resistant bacteria were isolated on colistin-supplemented CHROMagar Orientation plates, and the species were identified by matrix-assisted laser desorption/ionization time-of-flight assay. The presence of mcr genes was confirmed by polymerase chain reaction analysis. We examined the minimum inhibitory concentrations (MICs) of 16 antimicrobial agents against the isolates using agar diffusion and broth microdilution methods. Whole-genome sequencing (WGS) was used to explore the molecular characteristics and relationships of mcr-1-positive Escherichia coli (MCRPEC). Results Overall, 143 (57.2%) colistin-resistant bacteria were isolated, of which, 56 (22.4%, including 54 Escherichia coli and two Klebsiella pneumoniae) and four Aeromonas species were positive for mcr-1 and mcr-3, respectively. The animal-derived MCRPEC were significantly more prevalent in integrated fishery samples (40.0%) than those in market (4.8%, P 90%) but were susceptible to carbapenems and tigecycline. WGS analysis suggested that mcr-1 was mainly contained on plasmids, including IncHI2 (29.6%), IncI2 (27.8%), IncX4 (14.8%), and IncP (11.1%). Genomic analysis suggested mcr-1 transmission via the aquatic food chain. Conclusions MCRPEC were highly prevalent in the aquaculture supply chain, with the isolates showing resistance to most antibiotics. The data suggested mcr-1 could be transferred to humans via the aquatic food chain. Taking the “One Health” perspective, aquaculture should be incorporated into systematic surveillance programs with animal, human, and environmental monitoring

Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation

Multiplex PCR for detection of MCR genes in clinical fecal samples

Plasmid-mediated colistin-resistance genes have been reported worldwide in recent years. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect transferable colistinresistance genes (mcr-1 to mcr-6) in Enterobacteria for clinical laboratory purposes.The authors first designed six new primer pairs to amplify mcr-1 to mcr-6 gene products to achieve stepwise separation of amplicons between 87 to 216 bp,then divided these primers into two subgroups with the assistance of a pair of universal primers for the detection of currently described mcr genes and their variants in Enterobacteria. The protocol was validated by testing 29 clinical isolates of Escherichia coli of human origin, each well characterised and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. It was able to detect mcr-3 and mcr-4 as singletons or in combination. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance in limited resources conditions, and this method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance

Dietary Factors of blaNDM Carriage in Health Community Population: A Cross-Sectional Study

Aim: There is an ongoing debate as to what extent antimicrobial resistance (AMR) can be transmitted from dietary to humans via the consumption of food products. We investigated this association between dietary and global spreading carbapenem-resistant gene blaNDM Methods: We did a cross-sectional study to assess the risk factors for carrier of blaNDM in health community. Healthy adults were recruited from the residents attending Community Healthcare Service in Shenzhen City (Guangdong Province, China), through 1February 2018 to 31December 2019, and 718 pre-participants were included in this study. Questionnaire were obtained and the qualitative food frequency questionnaire (Q-FFQ) were used to assess dietary intake. qPCR was applied to confirm the carrier of blaNDM in participants’fecal samples. Multivariable logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (95% CI) of each outcome according to each dietary factor before and after prosperity score matching (PSM). Results: we showed that a high intake of coarse grain (OR 1.003; 95% CI 1.001–1.005, p < 0.01) and root and tuber crops (OR 1.003; 95% CI 1.001–1.004, p < 0.05) were independent risk factor for blaNDM carrier in health communities, suggesting a possible transfer of AMRbetweendietary andhumans. Surprisingly, we also showed an association between a higher intake of poultry as a protective, which may be explained by the beneficial effects on the gut microbiota. Conclusion: Dietary factors such as intake of coarse grain, root and tuber crops and poultry were associated with blaNDM carrier in health communities. The influence of dietary factorson blaNDM carrier in the present study provides insights for the tangible dietary advice with guidelines to the routine of people with the risk of blaNDM carrier. This demonstrates the role of dietary intake in the prevention of blaNDM carrier, since prevention is the best way to control modifiable risk factors. A lower carrier rate of blaNDM is helpful to reduce the possibility of transmission and pathogenicity. Further studies on food, microbiota and antimicrobial resistance are necessary to confirm this possible association and unravel underlying mechanisms

Novel Variant of New Delhi Metallo-β-lactamase, NDM-20, in Escherichia coli

The spread of carbapenem-resistant Enterobacteriaceae (CRE) mediated by New Delhi metallo-β-lactamase (NDM) poses a serious challenge to clinicians and has become a major public health concern. NDM has been evolving into variants that possess different hydrolysis activity toward antibiotics, so as to affect treatment strategy. In addition, very few studies on NDM variants have focused on animal-derived bacterial isolates. Our study reports a novel NDM variant, NDM-20, in an isolate of Escherichia coli CCD1 recovered from the food animal swine in China. The isolate that was assigned to ST1114, exhibited high level resistance to all β-lactams tested, including aztreonam and carbapenems. The gene of blaNDM-20 was located on an IncX3-type plasmid, surrounded by multiple insertion sequences. Sequencing analysis demonstrated that blaNDM-20 contained three point mutations at positions 262 (G→T), 460 (A→C), and 809 (G→A), compared with blaNDM-1, and just one point mutation at position 809 (G→A), relative to blaNDM-5. Functional analysis revealed that the blaNDM-20 transformant, DH5α+pHSG398/NDM-20, exhibited a higher resistance to ertapenem than that of blaNDM-1 transformant DH5α+pHSG398/NDM-1. Kinetic parameter analysis showed that NDM-20 had increased enzymatic activity against some penicillins and cephalosporins but decreased carbapenemase activity relative to NDM-5. The identification of NDM-20 further confirms the evolution and prevalence of NDM variants in bacteria of food-animal origin

Protective Effects of Extracts from Fructus rhodomyrti against Oxidative DNA Damage In Vitro and In Vivo

Objective. To evaluate the potential protective effects of extracts from Fructus rhodomyrti (FR) against oxidative DNA damage using a cellular system and the antioxidant ability on potassium bromate- (KBrO3-) mediated oxidative stress in rats. Methods. The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3. Results. FR was shown to effectively protect against DNA damage induced by H2O2  in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats. Conclusions. Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent manner in vitro and in vivo

Synthesis of haptens and production of antibodies to bisphenol A

Three immunizing haptens of bisphenol A (BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) value of the best polyclonal antibody was 2.1 mg·L−1, based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible cross-reactivity with bisphenol B and bisphenol E. A sensitive icELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples (5, 10 and 20 mg·L−1), the recovery ranged from 80% to 102% with a coefficient of variation (CV) value below 15.8%. The limit of detection of icELISA was 1.95 mg·L−1. These results indicate that the icELISA method is suitable for the detection of BPA in milk

An Innovative Nanobody-Based High-Biocompatibility Gold Interdigitated Microelectrode Electrochemical Bioimpedance Sensor for the Ultrasensitive Detection of Difenacoum in Human Serum

Difenacoum (DIF) is one of the most widely used anticoagulant rodenticides. However, accidental or intentional ingestion of DIF seriously threatens humans and other non-target species. Therefore, a rapid and sensitive detection method to quantify DIF is urgently needed. In this study, one anti-DIF nanobody (Nb) was assembled on the surface of a gold interdigitated microelectrode (IDME) using an Au–S bond to fabricate a bioimpedance sensor. To improve the immobilization amount of Nbs on the electrode, a polycrystalline gold IDME was prepared to provide a larger surface and better biocompatibility. Thus, a novel and ultrasensitive bioimpedance sensor based on electrochemical impedance spectroscopy (EIS) was designed for the determination of DIF, and it displayed good reproducibility and stability in human serum. The proposed bioimpedance sensor displayed a wide working range, between 0.1–1000 pg/mL, with a limit of detection (LOD) of 0.1 pg/mL of DIF. This method exhibited excellent performance, good sensitivity, and reproducibility and achieved the highest sensitivity of all currently existing methods used to quantify DIF. The highly sensitive DIF detection of this proposed bioimpedance sensor indicates its potential as an efficacious approach for DIF monitoring in human serum with high accuracy and precision

Confirmatory Analysis of Nitroimidazoles and Hydroxy Metabolites in Honey by Dispersive-Solid Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

An ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS/MS) method was developed and validated for confirmatory analysis of four nitroimidazoles and three hydroxy metabolites in honey. Honey samples were dissolved in 2% formic acid solution and nitroimidazoles and metabolites were isolated and enriched by dispersive-solid phase extraction using mixed-mode strong cation-exchange sorbent. The determination involves separation of analytes on an UHPLC C18 column and detection by multiple reaction monitoring in positive ionization mode. The recovery of the method was ranged from 90.2 to 105.6% with inter-day relative standard deviations of less than 11.2%. The limits of detection and limits of quantification were in the ranges of 0.02⁻0.07 µg/kg and 0.05⁻0.2 µg/kg, respectively. Honey samples from the market were analyzed to demonstrate the applicability of the proposed method