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BmNPV infection induces nuclear vesicle-like structures and its Bm5 protein mediates the intranuclear lipid accumulation, which is thought to participate in the formation of nuclear vesicles. However, the relationship between viral-induced nuclear vesicles and Bm5 protein is still unclear. Here, our results indicated that BmNPV Bm5 protein participated in the baculovirus infection-induced nuclear vesicle-like structures' invagination thereby influencing the production of occlusion-derived virion (ODV) and occlusion body (OB). The process of nuclear vesicle-like structures' formation was dispensable for the transport or recruitment of ODV major envelope proteins, such as P74 and Bm14. Furthermore, baculovirus-induced nuclear F-actin might provide a direct mechanical force to mediate the scission of large vesicle-like structures from the nuclear membrane. Collectively, these findings illustrated a BmNPV Bm5 protein-induced nuclear membrane invagination pathway and revealed the function of nuclear vesicle-like structures in ODV production.

Insect midgut plays a central role in food digestion and nutrition absorption. Larval silkworm midgut could be divided into 3 distinct regions based on their morphological colors. However, it remains rudimentary of regional gene expression and physiological function in larval silkworm midgut. Through transcriptome sequencing of 3 midgut compartments, a comprehensive analysis of gene expression atlas along the anterior-posterior axis was conducted. Posterior midgut was found transcriptionally divergent from anterior and middle midgut. Differentially expressed gene analysis revealed the regional specialization of digestive enzyme production, transmembrane transport, chitin metabolism, and hormone regulation in different midgut regions. In addition, gene subsets of pan-midgut and region-specific transcription factors (TFs) along the length of midgut were also identified. The results suggested that homeobox TFs might play an essential role in transcriptional variations across the midgut. Altogether, our study provided the first fundamental resource to investigate physiological function and regulation mechanism in larval midgut compartmentalization.

Bloodstream infections (BSIs) caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are potentially life-threatening and an urgent threat to public health. The present study aims to clarify the characteristics of carbapenemase-encoding and virulent plasmids, and their interactions with the host bacterium. A total of 425 Kp isolates were collected from the blood of BSI patients from nine Chinese hospitals, between 2005 and 2019. Integrated epidemiological and genomic data showed that ST11 and ST307 Kp isolates were associated with nosocomial outbreak and transmission. Comparative analysis of 147 Kp genomes and 39 completely assembled chromosomes revealed extensive interruption of acrR by ISKpn26 in all Kp carbapenemase-2 (KPC-2)-producing ST11 Kp isolates, leading to activation of the AcrAB-Tolc multidrug efflux pump and a subsequent reduction in susceptibility to the last-resort antibiotic tigecycline and six other antibiotics. We described 29 KPC-2 plasmids showing diverse structures, two virulence plasmids in two KPC-2-producing Kp, and two novel multidrug-resistant (MDR)-virulent plasmids. This study revealed a multifactorial impact of KPC-2 plasmid on Kp, which may be associated with nosocomial dissemination of MDR isolates.

Protein signaling complexes play important roles in prevention of several cancer types and can be used for development of targeted therapy. The roles of signaling complexes of phosphodiesterase 3B (PDE3B) and Rap guanine nucleotide exchange factor 3 (RAPGEF3), which are two important enzymes of cyclic adenosine monophosphate (cAMP) metabolism, in cancer have not been fully explored. In the current study, a natural product Kaempferol-3-O-(3'',4''-di-E-p-coumaroyl)-α-L-rhamnopyranoside designated as KOLR was extracted from Cinnamomum pauciflorum Nees leaves. KOLR exhibited higher cytotoxic effects against BxCP-3 pancreatic cancer cell line. In BxPC-3 cells, the KOLR could enhance the formation of RAPGEF 3/ PDE3B protein complex to inhibit the activation of Rap-1 and PI3K-AKT pathway, thereby promoting cell apoptosis and inhibiting cell metastasis. Mutation of RAPGEF3 G557A or low expression of PDE3B inactivated the binding action of KOLR resulting in KOLR resistance. The findings of this study show that PDE3B/RAPGEF3 complex is a potential therapeutic cancer target.

Breast cancer is considered to be the most prevalent cancer in women worldwide, and metastasis is the primary cause of death. Protease-activated receptor 1 (PAR1) is a GPCR family member involved in the invasive and metastatic processes of cancer cells. However, the functions and underlying mechanisms of PAR1 in breast cancer remain unclear. In this study, we found that PAR1 is highly expressed in high invasive breast cancer cells, and predicts poor prognosis in ER-negative and high-grade breast cancer patients. Mechanistically, Twist transcriptionally induces PAR1 expression, leading to inhibition of Hippo pathway and activation of YAP/TAZ; Inhibition of PAR1 suppresses YAP/TAZ-induced epithelial-mesenchymal transition (EMT), invasion, migration, cancer stem cell (CSC)-like properties, tumor growth and metastasis of breast cancer cells in vitro and in vivo. These findings suggest that PAR1 acts as a direct transcriptionally target of Twist, can promote EMT, tumorigenicity and metastasis by controlling the Hippo pathway; this may lead to a potential therapeutic target for treating invasive breast cancer.

Resistance-nodulation-division (RND) efflux pumps are important determinants of multidrug resistance in Gram-negative bacteria. As one of the typical members of the RND superfamily, Escherichia coli OqxAB multidrug efflux pump confers resistance to antimicrobial agents, such as olaquindox and fluoroquinolone. In the present study, site-directed mutagenesis and antimicrobial susceptibility measurement assay were applied to identify the crucial residues within OqxB, the transporter component of the OqxAB efflux pump system. It was found that alanine substitution of proton translocation pathway residues D410, D411, and R976 resulted in a complete loss of the transport function. Further studies revealed that the charge property of these residues is important for proper function of OqxB. Alanine replacement of residues involved in substrate-binding domains, including V141, F180, Y330, and F626, exhibited different responses toward different antimicrobial agents. Conservative replacement of Y330, F626, and F180 with amino acids having similar aromatic ring structure resulted in full or partial recovery of the efflux function. Molecular docking analysis demonstrated that olaquindox may form hydrogen bonds with F626, Y330, and V141, whereas only Y330 and F180 may interact with ciprofloxacin, implicating the different roles played by these residues when transporting different kinds of substrates. Graphical Abstract [Figure: see text].

Summary: Background: The global dissemination of colistin resistance encoded by mcr-1 has been attributed to extensive use of colistin in livestock, threatening colistin efficacy in medicine. The emergence of mcr-1 in common pathogens, such as Escherichia coli, is of particular concern. China banned the use of colistin in animal feed from May 1, 2017. We investigated subsequent changes in mcr-1 prevalence in animals, humans, food, and the environment, and the genomic epidemiology of mcr-1-positive E coli (MCRPEC). Methods: Sampling was done before (October to December, 2016) and after (October to December, 2017, and 2018, respectively) the colistin ban. 3675 non-duplicate pig faecal samples were collected from 14 provinces (66 farms) in China to measure intervention-related changes in mcr-1 prevalence. 15 193 samples were collected from pigs, healthy human volunteers, patients colonised or infected with Enterobacteriaceae who were admitted to hospital, food and the environment in Guangzhou, to characterise source-specific mcr-1 prevalence and the wider ecological effect of the ban. From these samples, 688 MCRPEC were analysed with whole genome sequencing, plasmid conjugation, and S1 pulsed-field gel electrophoresis with Southern blots to characterise associated genomic changes. Findings: After the ban, mcr-1 prevalence decreased significantly in national pig farms, from 308 (45%) of 684 samples in 2016 to 274 (19%) of 1416 samples in 2018 (p

Background Bloodstream infection (BSI) caused by carbapenem-resistant Enterobacteriaceae are potentially life-threatening related to poorer outcomes. Colistin is considered one of the last-resort treatments against human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Therefore, emergence of strains from the blood that co-harboring mcr and carbapenem resistance genes were considered as a serious problem. Purpose In this study, two mcr-9-harboring MDR Enterobacter cloacae isolates BSI034 and BSI072 recovered from BSI patients were identified, one of which co-harbored mcr-9 and bla NDM-1. The genetic characteristics of the MDR plasmid needed to be clarified. Methods S1-PFGE and Southern blotting were conducted to determine the location of mcr-9. Whole-genome sequencing was performed to obtain the complete genome and plasmid sequences. The resistome and virulence genes of the strains, accompanied by the genetic characteristics of mcr-9- and bla NDM-1-harboring plasmids, were analyzed. Results Whole-genome sequencing showed that BSI034 harbored mcr-9-carrying IncHI2-type pBSI034-MCR9 and bla NDM-1-carrying IncX3-type pBSI034-NDM1. The 278,517 bp pBSI034-MCR9 carried mcr-9 along with the other 19 resistance genes. mcr-9 was flanked by IS903B (1057 bp) and IS26 (820 bp) in the same orientation. In addition to resistance genes, strain BSI034 also carried a chromosome-located Yersinia high-pathogenicity island, which harbored genes of yersiniabactin biosynthesis operon ybtSXQPAUTE, irp1/2, and fyuA. Conclusion We described the complete genome and mcr-9/bla NDM-1-co-harboring plasmid of E. cloacae from a BSI patient. Notable differences were observed within mosaic modules between pBSI034-MCR9 and other mcr-9-harboring plasmids due to extensive recombination via horizontal gene transfer.

Mycobacterium grossiae sp. nov. of type strain PB739T is a Gram-positive acid–alcohol–fast rod-shaped bacterium, which was recently isolated from a 76-year-old male who suffered from a 1-year history of hemoptysis. This strain was described as novel species in Mycobacterium genus. In this study, its genome was completely sequenced by PacBio technology, analyzed, and compared with other selected complete genome sequences of Mycobacterium to elucidate the distinct pathogenic features of the strain. The genomic analysis revealed that the genome of PB739T consists of one circular DNA chromosome of 5,637,923 bp with a GC content of 70.48% and one plasmid of 43,679 bp with a GC content of 66.24%. The entire genome contains 5434 predicted coding genes, 48 tRNAs, and 6 rRNA genes. Genome and comparative genomics against M. grossiae SCH identified three tandem short-chain dehydrogenase (SDR) genes which only exist in PB739T. These three tandem SDR genes locate in a Genomic island which was identified by Island Viewer. These SDR genes were predicted to be horizontally transferred from a Streptomyces ancestor based on phylogeny. Analysis of the mutant ΔSDR confirmed the relationship between these tandem genes with biofilm and pathogenicity. This report will provide us with an extended understanding of M. grossiae at the genomic level and would be helpful for understanding the evolution of Mycobacterium genus.

Lipases play crucial roles in food digestion by degrading dietary lipids into free fatty acids and glycerols. The domesticated silkworm (Bombyx mori) has been widely used as an important Lepidopteran model for decades. However, little is known about the lipase gene family in the silkworm, especially their hydrolytic activities as digestive enzymes. In this study, a total of 38 lipase genes were identified in the silkworm genome. Phylogenetic analysis indicated that they were divided into three major groups. Twelve lipases were confirmed to be expressed in the midgut at both transcriptional and translational levels. They were grouped into the same gene cluster, suggesting that they could have similar physiological functions. Quantitative real-time PCR (qRT-PCR) analyses indicated that lipases were mainly expressed in anterior and middle midgut regions, and their expression levels varied greatly along the length of midgut. A majority of lipases were down-regulated in the midgut when larvae stopped feeding. However, a unique lipase gene (Bmlip10583) showed low expression level during feeding stage, but it was significantly up-regulated during the larvae-pupae transition. These results demonstrated that expression of silkworm lipases was spatially and temporally regulated in the midgut during larval development. Taken together, our results provide a fundamental research of the lipase gene family in the silkworm.

Background Protein signaling complexes play important roles in prevention of several cancer types and can be used for development of targeted therapy. The roles of signaling complexes of phosphodiesterase 3B (PDE3B) and Rap guanine nucleotide exchange factor 3 (RAPGEF3), which are two important enzymes of cyclic adenosine monophosphate (cAMP) metabolism, in cancer have not been fully explored. Methods The natural product Kaempferol-3-O-(3′′,4′′-di-E-p-coumaroyl)-α-L-rhamnopyranoside designated as KOLR was extracted from Cinnamomum pauciflorum Nees leaves using reverse phase chromatography, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. The antitumor effect of KOLR was analyzed by multiple cell proliferation and metastasis experiments. The PDE3B/RAPGEF3 complex was found to be the target of KOLR through mRNA sequencing, Co-Immunoprecipitation assay, gene knock-down, gene mutation of drug-resistance cell line, and molecular docking. In vivo studies have shown that KOLR has the same antitumor mechanism. Results KOLR exhibited cytotoxic effects against selected cancer cells, except for AsPC-1 pancreatic cancer cell line. KOLR stabilized PDE3B/RAPGEF3 signaling complex thus inhibiting AKT phosphorylation and Rap-1 activation. Notably, mutation of RAPGEF3 G557A inhibited effect of KOLR on stabilizing PDE3B/RAPGEF3 complex in AsPC-1 cells. Furthermore, downregulation of PDE3B expression inhibited cytotoxic effect of KOLR on tumor cells. Downregulation of RAPGEF3 and Rap-1 expression promoted apoptosis of tumor cells and inhibited tumor metastasis. PDE3B inhibits activity of RAPGEF3 and activation of downstream signaling pathway. Conclusion The findings of this study show that KOLR could stabilize PDE3B/RAPGEF3 signaling complex to play an anti-tumor role and the PDE3B/RAPGEF3 complex is a potential therapeutic cancer target.

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

Purpose The main objective of this study was to decipher the prevalence, antimicrobial resistance, major virulence genes and the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from different clinical sources in southern China. Materials and methods The present study was performed on 187 non-duplicate S. aureus clinical isolates collected from three tertiary hospitals in Guangdong Province, China, 2010-2016. Antimicrobial susceptibility testing was performed by the disk diffusion method and by measuring the minimum inhibitory concentration. Screening for resistance and virulence genes was performed. Clonal relatedness was determined using various molecular typing methods such as multilocus sequence typing, spa and staphylococcal chromosomal cassette mec (SCCmec) typing. Whole genome sequencing was performed for three selected isolates. Results Out of 187 isolates, 103 (55%) were identified as MRSA. The highest prevalence rate was found among the skin and soft tissue infection (SSTI) samples (58/103), followed by sputum samples (25/103), blood stream infection samples (15/103) and others (5/103). Antimicrobial susceptibility results revealed high resistance rates for erythromycin (64.1%), clindamycin (48.5%), gentamicin (36.9%) and ciprofloxacin (33.98%). All isolates were susceptible to vancomycin. Resistance genes and mutation detected were as follows: aac(6')-aph(2") (24.3%), dfrG (10.7%), rpoB (21.4%), cfr (0%), fexA (1.94%), gyrA (35.92%), gyrB (0.97%), grlA (20.4%), grlB (10.68%), ermA (21.4%), ermB (18.44%), ermC (21.4%) and lnuA (18.44%). Profiling of virulence genes revealed the following: sea (11.7%), seb (21.4%), sec (0.97%), sed (0.97%), hla (86.41%), hlb (17.48%), hlg (10.68%), hld (53.4%), Tsst-1 (3.9%) and pvl (27.2%). Clonal relatedness showed that ST239-SCCmecA III-t37 clone was the most prevalent clone. Conclusion Our study elucidated the prevalence, antibiotic resistance, pathogenicity and molecular characteristics of MRSA isolated from various clinical sources in Guangdong, China. We found that the infectious rate of MRSA was higher among SSTI than other sources. The most predominant genotype was ST239-SCCmecA III-t37 clone, indicating that ST239-t30 clone which was previously predominant had been replaced by a new clone.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid. IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and the analysis of the plasmids associated with CR-hypervirulent-KP (CR-HvKP) in Egypt are not presented. Therefore, we attempt to decipher the plasmids sequences, which are responsible for transferring the determinants of carbapenem-resistance, particularly the blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383, and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed using Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as CR-HvKP strain, it harboured four plasmids, namely; pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes, blaNDM-1 and blaKPC-2, were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD, iutA). Thus, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We reported for the first time a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient from the ICU in a tertiary-care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid.IMPORTANCECRKP had been registered in the critical priority tier by the World Health Organization and became a significant menace to public health. Therefore, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. Herein, we reported for the first time (to the best of our knowledge) a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient in a tertiary-care hospital from the ICU in Egypt, which showed the cohabitation of a novel hybrid plasmid co-harbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid. Herein, the high rate of CRKP might be due to the continuous usage of carbapenems as empirical therapy, besides the failure to implement an antibiotic stewardship program in Egyptian hospitals. Thus, this study serves to alert the contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Two multidrug-resistant (MDR) mcr-1 -harboring Klebsiella pneumoniae isolates from patients with urinary tract infections and one MDR Klebsiella quasipneumoniae isolate from a patient with bloodstream infection were identified to carry tmexCD1-toprJ1 . The addition of the efflux pump inhibitor reduced the tigecycline MIC against all three isolates by 8- to 16-fold. pKQBSI104-1 was transferred from K. quasipneumoniae to Escherichia coli J53 via conjugation.

Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disorder in the colon and rectum leading to low life-quality and high societal costs. Ursolic acid (UA) is a natural product with pharmacological and biological activities. The studies are aimed at investigating the protective and treatment effects of UA against the dextran sulfate sodium- (DSS-) induced UC mouse model and its underlying mechanism. UA was orally administered at different time points before and after the DSS-induced model. Mice body weight, colon length, and histological analysis were used to evaluate colon tissue damage and therapeutic evaluation. Intestinal transcriptome and microbe 16 s sequencing was used to analyze the mechanisms of UA in the prevention and treatment of UC. The early prevention effect of UA could effectively delay mouse weight loss and colon length shorten. UA alleviated UC inflammation and lowered serum and colon IL-6 levels. Three classical inflammatory pathways: MAPKs, IL-6/STAT3, and PI3K were downregulated by UA treatment. The proportion of macrophages and neutrophils in inflammatory cell infiltration was reduced in UA treatment groups. UA could significantly reduce the richness of intestinal flora to avoid the inflammatory response due to the destruction of the intestinal epithelial barrier. The function of UA against UC was through reducing intestinal flora abundance and regulating inflammatory and fatty acid metabolism signaling pathways to affect immune cell infiltration and cytokine expression.

Background Bloodstream infection (BSI) caused by carbapenem-resistant Enterobacteriaceae are potentially life-threatening related to poorer outcomes. Colistin is considered one of the last-resort treatments against human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Therefore, emergence of strains from the blood that co-harboring mcr and carbapenem resistance genes were considered as a serious problem. Purpose In this study, two mcr-9-harboring MDR Enterobacter cloacae isolates BSI034 and BSI072 recovered from BSI patients were identified, one of which co-harbored mcr-9 and blaNDM-1. The genetic characteristics of the MDR plasmid needed to be clarified. Methods S1-PFGE and Southern blotting were conducted to determine the location of mcr-9. Whole-genome sequencing was performed to obtain the complete genome and plasmid sequences. The resistome and virulence genes of the strains, accompanied by the genetic characteristics of mcr-9- and blaNDM-1-harboring plasmids, were analyzed. Results Whole-genome sequencing showed that BSI034 harbored mcr-9-carrying IncHI2-type pBSI034-MCR9 and blaNDM-1-carrying IncX3-type pBSI034-NDM1. The 278,517 bp pBSI034-MCR9 carried mcr-9 along with the other 19 resistance genes. mcr-9 was flanked by IS903B (1057 bp) and IS26 (820 bp) in the same orientation. In addition to resistance genes, strain BSI034 also carried a chromosome-located Yersinia high-pathogenicity island, which harbored genes of yersiniabactin biosynthesis operon ybtSXQPAUTE, irp1/2, and fyuA. Conclusion We described the complete genome and mcr-9/blaNDM-1-co-harboring plasmid of E. cloacae from a BSI patient. Notable differences were observed within mosaic modules between pBSI034-MCR9 and other mcr-9-harboring plasmids due to extensive recombination via horizontal gene transfer.

BackgroundThe global dissemination of colistin resistance encoded by mcr-1 has been attributed to extensive use of colistin in livestock, threatening colistin efficacy in medicine. The emergence of mcr-1 in common pathogens, such as Escherichia coli, is of particular concern. Therefore, China banned the use of colistin in animal feed from May 1ST 2017. We investigated subsequent changes in mcr-1 prevalence, and the genomic epidemiology of mcr-1-positive Escherichia coli (MCRPEC).MethodsSampling was conducted pre- (October-December 2016) and post-colistin ban (October-December, 2017 and 2018, respectively). 3675 non-duplicate pig fecal samples were collected from 14 provinces (66 farms) in China to determine intervention-related changes in mcr-1 prevalence. 15193 samples were collected from pigs, healthy human volunteers, colonized and infected hospital inpatients, food and the environment in Guangzhou, to characterize source-specific mcr-1 prevalence and the wider ecological impact of the ban. From these samples, 688 MCRPEC were analyzed with whole genome sequencing (WGS), plasmid conjugation and S1-PFGE/Southern blots to characterize associated genomic changes.FindingsAfter the ban, mcr-1 prevalence decreased significantly in national pig farms, from 45·0% (308/684 samples) in 2016, to 19·4% (274/1416) in 2018 (pmcr-1 was predominantly found on plasmids (632/688 [92%]). Common mcr-1 plasmid types included IncX4, IncI2 and IncHI2 (502/656 [76.5%]); significant increases in IncI2-associated mcr-1 and a distinct lineage of mcr-1-associated IncHI2 were observed post-ban. Changes in the frequency of mcr-1-associated flanking sequences (ISApl1-negative MCRPEC), 63 core genome SNPs and 30 accessory genes were also significantly different after the ban, consistent with rapid genetic adaptation in response to changing selection pressures.InterpretationA rapid, ecosystem-wide, decline in mcr-1 was observed after banning the use of colistin in animal feed, with associated genetic changes in MCRPEC. Genomic surveillance is key to assessing and monitoring stewardship interventions.FundingNational Natural Science Foundation of China

One of the amazing phenomena in the baculovirus life cycle is the hyperexpression of the very late gene, polyhedrin (polh), causing the production of the occlusion bodies where progeny virions are embedded. However, to date, the molecular mechanism underlying its hyperexpression is not completely elucidated. Considering that, in this review, the mechanism responsible for its hyperexpression from the previous studies up to now was comprehensively summarized from three aspects, namely, the structure characteristics of the polh promoter and transcription regulation, the structure and translation regulation of the polh mRNA, and especially the regulators that influence the expression of polh gene. Moreover, this review will help us obtain a better understanding about the hyperexpression of polh, and also provide guidance for improving the expression efficiency of the foreign proteins by adopting the baculovirus expression vector system.

Resistance-nodulation-division (RND) efflux pumps are important determinants of multidrug resistance in Gram-negative bacteria. As one of the typical members of the RND superfamily

Mycobacterium grossiae sp. nov. of type strain PB739

Aiming at the unreasonable weight allocation in traditional teaching evaluation, rough set theory (RST) is applied to the process of teaching evaluation.Using the Teaching Quality evaluation data of Business English Majors from Guangxi Talent International College, we establish the evaluation information system, and calculate the importance of evaluation index, based on which, the weight of index is calculated. Finally, the evaluation results are calculated according to the weight of index. The weight of rough set index is determined entirely by the data itself, avoiding the influence of subjective factors, so it is more scientific, reasonable, objective and fair.

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

Purpose The main objective of this study was to decipher the prevalence, antimicrobial resistance, major virulence genes and the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from different clinical sources in southern China. Materials and methods The present study was performed on 187 non-duplicate S. aureus clinical isolates collected from three tertiary hospitals in Guangdong Province, China, 2010–2016. Antimicrobial susceptibility testing was performed by the disk diffusion method and by measuring the minimum inhibitory concentration. Screening for resistance and virulence genes was performed. Clonal relatedness was determined using various molecular typing methods such as multilocus sequence typing, spa and staphylococcal chromosomal cassette mec (SCCmec) typing. Whole genome sequencing was performed for three selected isolates. Results Out of 187 isolates, 103 (55%) were identified as MRSA. The highest prevalence rate was found among the skin and soft tissue infection (SSTI) samples (58/103), followed by sputum samples (25/103), blood stream infection samples (15/103) and others (5/103). Antimicrobial susceptibility results revealed high resistance rates for erythromycin (64.1%), clindamycin (48.5%), gentamicin (36.9%) and ciprofloxacin (33.98%). All isolates were susceptible to vancomycin. Resistance genes and mutation detected were as follows: aac(6’)-aph(2”) (24.3%), dfrG (10.7%), rpoB (21.4%), cfr (0%), fexA (1.94%), gyrA (35.92%), gyrB (0.97%), grlA (20.4%), grlB (10.68%), ermA (21.4%), ermB (18.44%), ermC (21.4%) and lnuA (18.44%). Profiling of virulence genes revealed the following: sea (11.7%), seb (21.4%), sec (0.97%), sed (0.97%), hla (86.41%), hlb (17.48%), hlg (10.68%), hld (53.4%), Tsst-1 (3.9%) and pvl (27.2%). Clonal relatedness showed that ST239-SCCmecA III-t37 clone was the most prevalent clone. Conclusion Our study elucidated the prevalence, antibiotic resistance, pathogenicity and molecular characteristics of MRSA isolated from various clinical sources in Guangdong, China. We found that the infectious rate of MRSA was higher among SSTI than other sources. The most predominant genotype was ST239-SCCmecA III-t37 clone, indicating that ST239-t30 clone which was previously predominant had been replaced by a new clone.

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5′ or/and 3′ flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super “chromatin opening element” and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content.

Glutathione S-transferase (GST) protein in gastrointestinal (GI) tracts of 16 organ donors, from whom all or substantial portions of the GI tract (stomach–colon) were available, was quantitated by HPLC and examined for interindividual variability/consistency of organ-specific patterns of expression. GSTP1, GSTA1, and GSTA2 were major components, and GSTM1 and GSTM3 were minor components. Consistent patterns of organ-specific expression were evident despite a high degree of interindividual variation of expression. GSTP1 was expressed throughout the GI tract and showed a decrease of expression from stomach to colon. GSTA1 and GSTA2 were expressed at high levels in duodenum and small intestine and expression decreased from proximal to distal small intestine. In contrast, GSTA1 and GSTA2 expression in colon and stomach of all subjects was low, particularly for colon where GSTA1 expression was 20- to 800-fold lower than that in corresponding small intestine. These consistent patterns of expression would suggest that compared to duodenum and small intestine, colon and to a lesser extent stomach always have low potential for GST-dependent detoxification of chemical carcinogens and are therefore at greater risk of genotoxic effects, particularly via substrates that are specific for GSTA1. This may be a factor in the greater susceptibility of stomach and colon to cancers compared to duodenum/small intestine.