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The emergence of the plasmid-borne colistin-resistant gene (mcr-1) poses a great threat to human health. What is worse, the recent observations of the co-existence of mcr-1 with other antimicrobial resistance genes in some bacteria cause further concern. Here, we present the first report of a wild Escherichia coli strain that co-carries an mcr-1 encoding phage-like IncY plasmid (pR15_MCR-1) and a bla NDM-5 encoding IncX3 plasmid (pR15_NDM-5) from a pharmaceutical industry, wastewater treatment plant, in China. This study highlights the spreading of E. coli carrying both mcr-1 and bla NDM-5 genes in the pharmaceutical industry. Importance Escherichia coli strains that carry both mcr-1 and bla NDM-5 genes are of great health concern and are already found in humans and animals worldwide, yet there is a paucity of observations of this resistant strain in the environment. Here we present the first isolation of an E. coli strain (R15) that co-carries mcr-1 and bla NDM-5 genes from a wastewater treatment plant in China. Whole-genome sequencing indicated that R15 harbored two plasmids, pR15_MCR-1 and pR15_NDM-5, that carry mcr-1 and bla NDM-5, respectively. The observation of this wild-derived E. coli strain that carries mcr-1 and bla NDM-5 genes simultaneously calls for the urgency to improve monitoring and reducing its further spreading.

A multidrug-resistant Citrobacter freundii strain R17 was isolated from a wastewater treatment plant in China. Whole-genome sequencing of strain R17 revealed a new sequence type (ST412) chromosome (length 5,124,258 bp) and an Inc FII (Yp) group plasmid pCFR17_1 (length 206,820 bp). A total of 13 antibiotic-resistance genes (ARGs) that confer resistance to eight different antibiotic groups were encoded by strain R17 and 12 of them were carried by plasmid pCFR17_1. These data and analysis suggest that the environment-derived C. freundii strains may serve as potential sources of ARGs and highlight the need of further surveillance of this bacteria in the future.

Metallo-β-lactamases (MBLs) are a group of enzymes that can inactivate most commonly used β-lactam-based antibiotics. Among MBLs, New Delhi metallo-β-lactamase-1 (NDM-1) constitutes an urgent threat to public health as evidenced by its success in rapidly disseminating worldwide since its first discovery. Here we report the biochemical and genetic characteristics of a novel MBL, ElBla2, from the marine bacterium Erythrobacter litoralis HTCC 2594. This enzyme has a higher amino acid sequence similarity to NDM-1 (56%) than any previously reported MBL. Enzymatic assays and secondary structure alignment also confirmed the high similarity between these two enzymes. Whole genome comparison of four Erythrobacter species showed that genes located upstream and downstream of elbla2 were highly conserved, which may indicate that elbla2 was lost during evolution. Furthermore, we predicted two prophages, 13 genomic islands and 25 open reading frames related to insertion sequences in the genome of E. litoralis HTCC 2594. However, unlike NDM-1, the chromosome encoded ElBla2 did not locate in or near these mobile genetic elements, indicating that it cannot transfer between strains. Finally, following our phylogenetic analysis, we suggest a reclassification of E. litoralis HTCC 2594 as a novel species: Erythrobacter sp. HTCC 2594.

Copper mine tailings are causing great environmental concern nowadays due to their high contents of heavy metals. These hazards may release to air, water, and soil, posing great threat to the living organisms in the surroundings. In the present work, we profiled the heavy metal contents, microbiome and resistome of a mine tailing in Dexing Copper Mine, which is the largest open-pit copper mine in China. A total of 39.75 Gb clean data was generated by metagenomics sequencing and taxonomy analysis revealed Actinobacteria, Proteobacteria, Acidobacteria, Euryarchaeota, and Nitrospirae as the most abundant phylum in this tailing. In general, 76 heavy metal resistance genes (HMRGs) and 194 antimicrobial resistance genes (ARGs) were identified with merA and rpoB2 as the most abundant HMRG and ARG, respectively. We also compared the differences of heavy metal concentrations among the six sampling sites in the same tailing and found that significant differences exited in copper and zinc. Hierarchical cluster analysis showed that the samples from the six sampling sites were clustering in two groups based on heavy metal concentrations. Accordingly, clustering based on microbial composition and relative abundances of resistance genes exhibited the same clustering pattern, indicating a possible shaping influence of heavy metals on the microbiome and resistome in this tailing. Our work presented heavy metal contents, microbial composition and resistance genes in a copper mine tailing of the largest copper mine in China, and these data will of great use in the surveillance, maintenance, and remediation of this tailing.

Background and objectives: Wastewater treatment plants (WWTPs) are one of the major reservoirs for antimicrobial resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in the environment. Thus, the investigation on ARB and ARGs from WWTPs has attracted increasing attention in recent years. In order to uncover the resistome in a WWTP treating effluents from a pharmaceutical industry in China, the extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains were isolated and their whole genome sequences were obtained and analyzed. Moreover, metagenomic sequencing was applied to give a comprehensive view of antibiotic resistance in this WWTP. Methods: 18 ESBL-producing E. coli strains were isolated from a WWTP located in Taizhou, China on April, 2017. All strains were sequenced using Illumina HiSeq 2000 sequencer. The whole genome sequences were assembled using SPAdes software and annotated with RAST server. Sequence types (STs), plasmids, ARGs and virulence genes were predicted from the genomes using MLST, Plasmid Finder, ResFinder and Virulence Finder, respectively. Metagenomic DNA of the same sample was extracted and sequenced using Illumina Hiseq X Ten platform. Metagenomic sequences were assembled using SOAPdenovo software. Results: All 18 ESBL-producing E. coli strains were resistant to ampicillin, cefazolin and ceftriaxone. Analysis of their genomes revealed that all strains carried beta-lactamase encoding genes and the most prevalent type was blaCTX-M. Various virulence genes and ARGs confronting resistance to other types of antimicrobial agents were also predicted. Further investigation on the metagenomics data indicated 11 ARGs with high amino acid identities to the known ARGs. Five of these ARGs, aadA1, aac(6’)-lb-cr, flo(R), sul2 and sul1, were also present in the genomes of the ESBL-producing E.coli isolated from the same sample. Conclusions: Our study revealed the resistome of a pharmaceutical WWTP by both culture-dependent and metegenomic methods. The existence of ESBL-producing E. coli strains, indicating that pharmaceutical WWTP can play a significant role in the emergence of ARB. The occurrence of ARGs annotated from the metagenomic data suggests that pharmaceutical WWTP can play a significant role in the emergence of ARGs. Our findings highlight the need for strengthening the active surveillance of ARB and ARGs from pharmaceutical industry.

Information on the weathering behaviour of Cunninghamia lanceolata (Lamb.) Hook. is needed to provide references for wood weatherproof pre-treatment and to improve wood utilization. Therefore, this study was conducted to understand the variation in the intrinsic weathering behaviour of Cunninghamia lanceolata (Chinese fir) under natural conditions. Wood samples from 15 Cunninghamia lanceolata trees aged 26&ndash, 30 years old were used. The structural degradation and discoloration of wood surfaces before and after exposure were compared. The results show that the weathering behaviour of wood was weakened from heartwood to sapwood and enhanced from the bottom to the top. This study provided information for weatherability research and improved wood utilization of Cunninghamia lanceolata.

Infections with Staphylococcus aureus, a common inducer of mastitis, often result in mammary gland damage and death of various cell types. Although S. aureus was suggested to induce apoptosis in a bovine mammary epithelial cell (BMEC) line, MAC-T, it is unknown whether primary BMECs (pBMECs) apoptosis is triggered by S. aureus and the associated underlying molecular mechanisms have not been determined. Here, we demonstrated that S. aureus induced apoptosis in pBMECs in a time- and dose-dependent manner. Further, S. aureus-induced apoptosis in pBMECs was associated with activation of caspase-3 and caspase-8, but caspase-9 was not. In addition, pBMECs apoptosis was mitigated by caspase-3 and caspase-8 inhibitors, suggesting that apoptosis is initiated via caspase-8 activation. Moreover, S. aureus infection significantly increased expressions of Fas and Fas-associated death domain (FADD) of pBMECs. Taken together, our results demonstrated that S. aureus induced apoptosis in pBMECs via the Fas-FADD death receptor and subsequently triggered the caspase-8-dependent signaling.

Staphylococcus aureus (S. aureus) is an important etiological organism in chronic and subclinical mastitis in lactating cows. Given the fundamental role the primary bovine mammary epithelial cells (pBMECs) play as a major first line of defense against invading pathogens, their interactions with S. aureus was hypothesized to be crucial to the establishment of the latter's infection process. This hypothesis was tested by investigating the global transcriptional responses of pBMECs to three S. aureus strains (S56,S178 and S36) with different virulent factors, using a tag-based high-throughput transcriptome sequencing technique. Approximately 4.9 million total sequence tags were obtained from each of the three S. aureus-infected libraries and the control library. Referenced to the control, 1720, 219, and 427 differentially expressed unique genes were identified in the pBMECs infected with S56, S178 and S36 S. aureus strains respectively. Gene ontology (GO) and pathway analysis of the S56-infected pBMECs referenced to those of the control revealed that the differentially expressed genes in S56-infected pBMECs were significantly involved in inflammatory response, cell signalling pathways and apoptosis. In the same vein, the clustered GO terms of the differentially expressed genes of the S178-infected pBMECs were found to comprise immune responses, metabolism transformation, and apoptosis, while those of the S36-infected pBMECs were primarily involved in cell cycle progression and immune responses. Furthermore, fundamental differences were observed in the levels of expression of immune-related genes in response to treatments with the three S. aureus strains. These differences were especially noted for the expression of important pro-inflammatory molecules, including IL-1α, TNF, EFNB1, IL-8, and EGR1. The transcriptional changes associated with cellular signaling and the inflammatory response in this study may reflect different immunomodulatory mechanisms that underlie the interaction between pBMECs and S. aureus strains during infection by the latter.

The mammalian target of rapamycin (mTOR) is an important controller of cell proliferation. RAD001 (everolimus) is a specific inhibitor of mTOR, can block mTOR signaling pathway. The purposes of this study are to explore the inhibitory effects of RAD001 on Cashmere goat fetal fibroblasts (GFb cells) proliferation. We examined both the effects of RAD001 on cell proliferation and cell shape of GFb cells. The GFb cells were treated with different concentrations of RAD001 (0.037 μM to 20.0μM) for 48h, and then the cell shape and proliferative density were observed with optical microscopy cell imaging. The results showed that GFb cells were sensitive to RAD001. RAD001 inhibits the cell proliferation. The morphological results indicated that mTOR is functional in GFb cells and acts as a key kinase to regulate cell proliferation. The synthesis of microfilament or organization of cytoskeleton perhaps was disrupted in GFb cells when mTOR was inhibited.

The mammalian target of rapamycin (mTOR) is an important controller of cell proliferation. RAD001 (everolimus) is a specific inhibitor of mTOR, can block mTOR signaling pathway. The purposes of this study are to explore the inhibitory effects of RAD001 on Cashmere goat fetal fibroblasts (GFb cells) proliferation. We examined both the effects of RAD001 on cell proliferation and cytostatic effect of GFb cells. The relative viability of the cells treated with RAD001 over 0.33 µM was significantly different compared with that of control (P < 0.01). As a result, RAD001 induced a dose-dependent decrease in cell proliferation. Taken together, these data showed that RAD001 can inhibit mTOR signaling and proliferation in GFb cells in vitro.