Genetic Diversity of Carbapenem-Resistant Enterobacteriaceae (CRE) Clinical Isolates From a Tertiary Hospital in Eastern China

The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is increasing globally, with different molecular mechanisms described. Here we studied the molecular mechanisms of carbapenem resistance, including clonal and plasmid dissemination, of 67 CRE isolates collected between 2012 and 2016 from a tertiary hospital in Eastern China, an CRE endemic region. Species identification and susceptibility testing were performed using the BD Phoenix Automated Microbiology System. Isolates were characterized by PCR (for carbapenemases, ESBLs, AmpC and porin genes), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and conjugation transfer experiments. Selected blaKPC-2 -harboring plasmids were subjected to next-generation sequencing using the Illumina Miseq platform. Among the 67 CRE isolates, 42 Klebsiella pneumoniae, 10 Serratia marcescens, 6 Enterobacter cloacae, 2 Raoultella ornithinolytica, 2 K. oxytoca, 1 K. aerogenes, and 4 Escherichia coli isolates were identified. Six different carbapenemases were detected, including blaKPC-2 (n = 45), blaKPC-3 (n = 1), blaNDM-1 (n = 6), blaNDM-5 (n = 1), blaIMP-4 (n = 2), and blaVIM-1 (n = 2); blaOXA-48-like genes were not detected. One E. cloacae strain possessed both blaNDM-1 and blaKPC-3, while two E. cloacae isolates harbored blaNDM-1 and blaVIM-1. ESBLs (CTX-M, SHV, and TEM) and/or AmpC (CMY, DHA, and ACT/MIR) genes were also identified in 59 isolates, including 13 strains that lacked carbapenemases. Several insertions or stop codon mutations were found within porin genes of K. pneumoniae, E. coli and S. marcescens isolates, both with and without carbapenemases. The 42 K. pneumoniae isolates belonged to 12 different sequence types (ST), with ST11 being the most common, while the 6 E. cloacae isolates comprised 4 different STs. The 10 S. marcescens all shared the same PFGE pulsotype, suggestive of clonal spread. Complete plasmid sequencing and PCR screening revealed both intra-strain and inter-species spread of a common blaKPC-2-harboring plasmid in our hospital. Taken together, our study revealed extensive genetic diversity among CRE isolates form a single Chinese hospital. CRE isolates circulating in the hospital differ significantly in their species, STs, porin genes, carbapenemase genes, and their plasmid content, highlighting the complex dissemination of CRE in this endemic region

Structural and Predicted Functional Diversities of Bacterial Microbiome in Response to Sewage Sludge Amendment in Coastal Mudflat Soil

The study investigated the influence of sewage sludge application at rates of 0 (CK), 30 (ST), 75 (MT), and 150 (HT) t ha−1 to mudflats on bacterial community diversity and predicted functions using amplicon-based sequencing. Soils under sewage sludge treatments, especially the HT treatment, exhibited lower pH, salinity and higher nutrient contents (C, N, and P). Moreover, restructured bacterial communities with significantly higher diversities and distinct core and unique microbiomes were observed in all sewage sludge-amended soils as compared to the control. Specifically, core bacterial families, such as Hyphomicrobiaceae, Cytophagaceae, Pirellulaceae Microbacteriaceae, and Phyllobacteriaceae, were significantly enriched in sewage sludge-amended soils. In addition, sewage sludge amendment significantly improved predicted functional diversities of core microbiomes, with significantly higher accumulative relative abundances of functions related to carbon and nitrogen cycling processes compared to the unamended treatment. Correlation analyses showed that modified soil physicochemical properties were conducive for the improvement of diversities of bacterial communities and predicted functionalities. These outcomes demonstrated that sewage sludge amendment not only alleviated saline–sodic and nutrient deficiency conditions, but also restructured bacterial communities with higher diversities and versatile functions, which may be particularly important for the fertility formation and development of mudflat soils

Effects of Different Exogenous Organic Materials on Improving Soil Fertility in Coastal Saline-Alkali Soil

The coastal saline-alkali soil in eastern China is an important reserve arable land resource. Adding exogenous organic material is an effective way to improve soil fertility and promote the conversion of saline-alkali soil to agricultural soil. In this study, a field plot experiment was used to investigate the influences of different organic materials (vinegar residue, VR; sewage sludge, SS; vermicompost, VC) on the reduction in salinity-alkalinity barrier factors, the accumulation of soil organic carbon (SOC), and the improvement in soil fertility in saline-alkali soil. The results indicated that applying different types of exogenous organic materials reduced soil electrical conductivity (EC) and pH, promoted SOC accumulation, and increased the barley yield. With the same application rate, VR application was more beneficial in reducing soil EC and pH, accumulating SOC, and increasing barley yield compared to SS and VC applications. In particular, the barley yield with VR application was higher than that with SS and VC applications by 18.4% and 26.6% on average, respectively, during the two-year experiment. Correlation and path analysis revealed that the barley yield was significantly negatively correlated with soil barrier factors (EC and pH), but EC in SS and VC-treated soils had an indirect negative effect on barley yield, while EC in VR-treated soil had a direct negative effect (−2.24). In addition, the direct (−4.46) and indirect (5.39) contributions of SOC to barley yield were higher with VR than those with SS and VC, while the direct contribution of soil aggregate to barley yield was lower with VR than that with SS and VC. Therefore, compared with SS and VC applications, VR application led to a fast reduction in soil barrier factors and the rapid accumulation of SOC, which were more beneficial for increasing barley yields in saline-alkali soil

Differential Effects of Organic Ameliorants on the Reassembly of Bacterial Communities in Newly Amended Coastal Mudflat Salt-Affected Soil

Understanding the influences of organic ameliorants amendment on the soil microbiome is fundamental for the alleviation of environmental constraints in coastal mudflat salt-affected soils. However, how the compositional and structural diversity of the microbial community responds to different organic inputs, and key environmental determinants and relevant mechanisms driving soil microbiome reassembly in coastal agroecosystems have not been illustrated. In this study, field experiments were conducted to investigate the effectiveness and underlying mechanisms of sewage sludge (S) and sludge-based vermicompost (V) at different application amounts (0, 50, 100, and 200 t ha−1) on variations in the compositional and structural diversities of soil bacterial community in coastal mudflats. The underlying driving factors, including soil physicochemical and enzymatic properties, were determined to estimate their effects on soil bacterial community. Results show that both types and amounts of organic ameliorants exerted significant influence on the alterations of bacterial community diversities. Particularly, restructured bacterial communities with significantly higher bacterial populations, lower Shannon diversities, and distinct core and unique community profiles were observed in organic ameliorant-amended soils as compared to CK. The co-occurrence networks of bacterial core OTUs reveal that V exhibited more profound effects than those of S on the scale and interactions enhancement and stability reduction of soil bacterial guilds. Additionally, both S and V significantly alleviated environmental constraints with lower pH and salinity, but higher soil organic carbon (SOC) amounts and enzyme activities were observed in the amended soils. Moreover, the effects of S and V on soil amendment were enhanced with the increase in the application amounts of organic ameliorants. Redundancy analysis (RDA) reveals that environmental factors (e.g., pH, salinity, SOC, sucrase, urease, and phosphatase) were pivotal determinants, accounting for 78.05% of the total bacterial community variations in coastal salt-affected soils across different treatments

Effects of Vermicompost Application on Growth and Heavy Metal Uptake of Barley Grown in Mudflat Salt-Affected Soils

China is facing a shortage of arable land resources, and the mudflat salt-affected soil along the east coast of China is an important reserve arable land resource. In this study, we conducted a randomized field trial to investigate the effects of vermicompost application rate (0, 25, 50, 125, and 250 t ha−1) on barley growth and heavy metal accumulation in mudflat salt-affected soil. We found that vermicompost application decreased bulk density, electrical conductivity (EC), and pH of mudflat salt-affected soil while increasing its organic carbon, nitrogen, and phosphorus contents. With the increase in vermicompost application rate, the yield of grain and total biomass of barley plants increased. The yield of grain in the vermicompost application treatments of 25, 50, 125, and 250 t ha−1 increased by 66.0%, 226.0%, 340.0%, and 512.0%, respectively, relative to the control. In addition, the concentrations of heavy metals (Cd, Cr, Cu, and Zn) in mudflat salt-affected soil and barley plant increased as the vermicompost application rate increased

Effects of Vermicompost Application on Growth and Heavy Metal Uptake of Barley Grown in Mudflat Salt-Affected Soils

China is facing a shortage of arable land resources, and the mudflat salt-affected soil along the east coast of China is an important reserve arable land resource. In this study, we conducted a randomized field trial to investigate the effects of vermicompost application rate (0, 25, 50, 125, and 250 t ha−1) on barley growth and heavy metal accumulation in mudflat salt-affected soil. We found that vermicompost application decreased bulk density, electrical conductivity (EC), and pH of mudflat salt-affected soil while increasing its organic carbon, nitrogen, and phosphorus contents. With the increase in vermicompost application rate, the yield of grain and total biomass of barley plants increased. The yield of grain in the vermicompost application treatments of 25, 50, 125, and 250 t ha−1 increased by 66.0%, 226.0%, 340.0%, and 512.0%, respectively, relative to the control. In addition, the concentrations of heavy metals (Cd, Cr, Cu, and Zn) in mudflat salt-affected soil and barley plant increased as the vermicompost application rate increased

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

<div><p>Purpose</p><p>Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.</p><p>Methods</p><p>Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.</p><p>Results</p><p>Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.</p><p>Discussion</p><p>Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.</p></div

Cycloheximide blocked sodium butyrate induced endoplasmic reticulum stress in colorectal cancer cells.

<p>HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showed the expression of IRE-1a, BIP, PDI, and GADPH (loading control) in HCT-116 (A) or HT-29 (C) cells. Protein expression was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.</p

Cyclohexamide and mithramycin blocked sodium butyrate induced autophagy in colorectal cancer cells.

<p>HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (A-B) HCT-116 cells treated with cyclohexamide; (C-D) HCT-116 cells treated with mithramycin; (E-F) HT-29 cells treated with cyclohexamide; (G-H) HT-29 cells treated with mithramycin.</p