Sanghuangporus sanghuang extract inhibits the proliferation and invasion of lung cancer cells in vitro and in vivo.

BACKGROUND/OBJECTIVES: Sanghuangporus sanghuang (SS) has various medicinal effects, including anti-inflammation and anticancer activities. Despite the extensive research on SS, its molecular mechanisms of action on lung cancer are unclear. This study examined the impact of an SS alcohol extract (SAE) on lung cancer using in vitro and in vivo models. MATERIALS/METHODS: Different concentrations of SAE were used to culture lung cancer cells (A549 and H1650). A cell counting kit-8 assay was used to detect the survival ability of A549 and H1650 cells. A scratch assay and transwell cell invasion assay were used to detect the migration rate and invasive ability of SAE. Western blot analysis was used to detect the expression of B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax), cyclin D1, cyclin-dependent kinases 4 (CDK4), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3). Lung cancer xenograft mice were used to detect the inhibiting ability of SAE in vivo. Hematoxylin and eosin staining and immunohistochemistry were used to detect the effect of SAE on the structural changes to the tumor and the expression of Bcl-2, Bax, cyclin D1, CDK4, STAT3, and p-STAT3 in lung cancer xenograft mice. RESULTS: SAE could inhibit lung cancer proliferation significantly in vitro and in vivo without cytotoxicity. SAE suppressed the viability, migration, and invasion of lung cancer cells in a dose and time-dependent manner. The SAE treatment significantly decreased the proapoptotic Bcl-2/Bax ratio and the expression of pro-proliferative proteins Cyclin D1 and CDK4 in vitro and in vivo. Furthermore, SAE also inhibited STAT3 expression. CONCLUSIONS: SAE reduced the cell viability and suppressed cell migration and invasion in human lung cancer cells. Moreover, SAE also exhibited anti-proliferation effects in vivo. Therefore, SAE may have benefits in cancer therapy.

Genome-wide chromatin interaction profiling reveals a vital role of super-enhancers and rearrangements in host enhancer contacts during BmNPV infection.

As influential regulatory elements in the genome, enhancers control gene expression under specific cellular conditions, and such connections are dynamic under different conditions. However, because of the lack of a genome-wide enhancer-gene connection map, the roles and regulatory pattern of enhancers were poorly investigated in insects, and the dynamic changes of enhancer contacts and functions under different conditions remain elusive. Here, combining Hi-C, ATAC-seq, and H3K27ac ChIP-seq data, we generate the genome-wide enhancer-gene map of silkworm and identify super-enhancers with a role in regulating the expression of vital genes related to cell state maintenance through a sophisticated interaction network. Additionally, a radical attenuation of chromatin interactions is found after infection of Bombyx mori nucleopolyhedrovirus (BmNPV), the main pathogen of silkworm, which directly rearranges the enhancer contacts. Such a rearrangement disturbs the intrinsic enhancer-gene connections in several antiviral genes, resulting in reduced expression of these genes, which accelerates viral infection. Overall, our results reveal the regulatory role of super-enhancers and shed new light on the mechanisms and dynamic changes of the genome-wide enhancer regulatory network.

Comparative Genomic Analysis of Hypervirulence Carbapenem-Resistant Klebsiella pneumoniae from Inpatients with Infection and Gut Colonization, China.

Background: The emergence and spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a potential epidemiological threat that needs to be monitored. However, the transmission and pathogenic characteristics of hv-CRKP in China remain unclear. We investigated the epidemiological characteristics of gut colonized hv-CRKP in a hospital in Guangdong Province, China. Methods: A total of 46 gut colonized hv-CRKP isolates were collected from Sun Yat-Sen Memorial Hospital (Guangzhou, China) from August 31st to December 31st, 2021. Minimum inhibitory concentrations (MICs) were obtained for 15 antibiotics for 46 hv-CRKP isolates. BALB/C mice infection model and mucoviscosity assay was used to evaluate the virulence of the isolates. The characteristics of genome, phylogenetic relationship and the structure of the plasmid of 46 gut colonized hv-CRKP isolates were compared with pathogenic isolates from GeneBank based on whole-genome data. Results: The hv-CRKP isolation rate of all gut colonized carbapenem-resistant Klebsiella pneumoniae was 17% (46/270), and the intestinal colonization rate of hv-CRKP was irrelevant to the sex, age, department of hospitalization, and history of antibiotic use of the host. The gut colonized hv-CRKP showed pandrug resistance and hypervirulence. The gut colonized hv-CRKP and pathogenic hv-CRKP prevalent in China were mainly ST11 hv-CRKP and had two major epidemic clades. The similarities in genomic characteristics between gut colonized hv-CRKP and pathogenic hv-CRKP were consistent. The gut colonized hv-CRKP carried an incomplete structure pK2044 virulence plasmid from hypervirulent K. pneumoniae NTUH-K2044 by analyzing the virulence plasmid structure. Conclusion: Our results suggest that the gut colonized ST11 hv-CRKP may serve as a reservoir for the clinical pathogenic ST11 HV-CRKP. It is necessary to further strengthen the monitoring of gut colonized hv-CRKP and research the potential mechanism of infection caused by gut colonized hv-CRKP.

Identification and characterization of BmNPV Bm5 protein required for the formation of nuclear vesicle structures.

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.

Multi-stimuli responsive Cu-MOFs@Keratin drug delivery system for chemodynamic therapy.

Although the potential of metal-organic framework (MOF) nanoparticles as drug delivery systems (DDS) for cancer treatment has been established by numerous studies, their clinical applications are still limited due to relatively poor biocompatibility. We fabricated a multifunctional Cu-MOFs@Keratin DDS for loaded drug and chemodynamic therapy (CDT) against tumor cells. The Cu-MOFs core was prepared using a hydrothermal method, and then loaded with the anticancer drug DOX and wrapped in human hair keratin. The Cu-MOFs@Keratin was well characterized by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and X-ray photoelectron spectroscopy (XPS). Characterization and pharmacokinetic studies of Cu-MOFs@Keratin were performed in vitro and in vivo. The keratin shell reduced the cytotoxicity and potential leakage of Cu-MOFs to normal cells, and allowed the drug-loaded nanoparticles to accumulate in the tumor tissues through enhanced permeability and retention effect (EPR). The particles entered the tumor cells via endocytosis and disintegrated under the stimulation of intracellular environment, thereby releasing DOX in a controlled manner. In addition, the Cu-MOFs produced hydroxyl radicals (·OH) by consuming presence of high intracellular levels of glutathione (GSH) and H2O2, which decreased the viability of the tumor cells.

Overexpression Bombyx mori HEXIM1 Facilitates Immune Escape of Bombyx mori Nucleopolyhedrovirus by Suppressing BmRelish-Driven Immune Responses.

Bombyx mori nucleopolyhedrovirus (BmNPV), a typical arthropod-specific enveloped DNA virus, is one of the most serious pathogens in silkworm farming, but the potential mechanisms of the evasion of innate immune responses from BmNPV infection are still poorly understood. HEXIM1 is an RNA-binding protein, best known as an inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation by RNA polymerase II. In this study, Bombyx mori HEXIM1 (BmHEXIM1) was cloned and characterized, and its expression was found to be remarkably upregulated after BmNPV infection. Furthermore, BmHEXIM1 was detected to increase the proliferation of BmNPV, and its full length is essential for assisting BmNPV immune escape by suppressing BmRelish-driven immune responses. This study brought new insights into the mechanisms of immune escape of BmNPV and provided theoretical guidance for the breeding of BmNPV-resistant silkworm varieties.

Mechanistic insights on cytotoxicity of KOLR, Cinnamomum pauciflorum Nees leaf derived active ingredient, by targeting signaling complexes of phosphodiesterase 3B and rap guanine nucleotide exchange factor 3.

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.

BmNPV p35 Reduces the Accumulation of Virus-Derived siRNAs and Hinders the Function of siRNAs to Facilitate Viral Infection.

Antiviral immunity involves various mechanisms and responses, including the RNA interference (RNAi) pathway. During long-term coevolution, viruses have gained the ability to evade this defense by encoding viral suppressors of RNAi (VSRs). It was reported that p35 of baculovirus can inhibit cellular small interference RNA (siRNA) pathway; however, the molecular mechanisms underlying p35 as a VSR remain largely unclear. Here, we showed that p35 of Bombyx mori nucleopolyhedrovirus (BmNPV) reduces the accumulation of virus-derived siRNAs (vsiRNAs) mapped to a particular region in the viral genome, leading to an increased expression of the essential genes in this region, and revealed that p35 disrupts the function of siRNAs by preventing them from loading into Argonaute-2 (Ago2). This repressive effect on the cellular siRNA pathway enhances the replication of BmNPV. Thus, our findings illustrate for the first time the inhibitory mechanism of a baculovirus VSR and how this effect influences viral infection.

Comparative transcriptome analysis reveals regional specialization of gene expression in larval silkworm (Bombyx mori) midgut.

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.

Actin Contributes to the Hyperexpression of Baculovirus Polyhedrin (polh) and p10 as a Component of Transcription Initiation Complex (TIC).

Hyperexpression of polh and p10, two very late genes, is one of the remarkable characteristics in the baculovirus life cycle. However, the mechanisms underlying the hyperexpression of these two genes are still incompletely understood. In this study, actin was identified as a highly potential binding partner of polh and p10 promoters by conducting DNA pull-down and LC-MS/MS analyses. Inhibiting actin dynamics delayed and decreased the transcription of polh and p10. Actin interacted with viral RNA polymerase and transcription regulators, and the nuclear import of viral polymerase was inhibited with the disruption of actin dynamics. Simultaneously, the high enrichment of actin in polh and p10 promoters discovered via a chromatin immunoprecipitation (ChIP) assay indicated that actin was a component of the viral polymerase TIC. Moreover, overexpression of actin surprisingly upregulated the expression of luciferase (Luc) under the control of polh and p10 promoters. Taken together, actin participated in the hyperexpression of polh and p10 as a component of TIC. These results facilitate the promotion of the expression efficiency of foreign genes in the baculovirus expression vector system (BEVS).

Molecular mechanism responsible for the hyperexpression of baculovirus polyhedrin.

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.

Genome-wide identification of lipases in silkworm (Bombyx mori) and their spatio-temporal expression in larval midgut.

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.

Ursolic Acid Regulates Intestinal Microbiota and Inflammatory Cell Infiltration to Prevent Ulcerative Colitis.

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.

Emergence of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Coharboring a blaNDM-1-Carrying Virulent Plasmid and a blaKPC-2-Carrying Plasmid in an Egyptian Hospital.

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.

Molecular characterization of carbapenem-resistant and virulent plasmids in Klebsiella pneumoniae from patients with bloodstream infections in China.

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.

Characterization of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump in Klebsiella pneumoniae and Klebsiella quasipneumoniae from Patients in China.

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. The tmexCD1-toprJ1-carrying plasmids pKP15ZE495-1 (102,569 bp) and pKQBSI104-1 (121,996 bp) were completely sequenced and analyzed.

Twist-mediated PAR1 induction is required for breast cancer progression and metastasis by inhibiting Hippo pathway.

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.

Co-Occurrence of mcr-9 and bla NDM-1 in Enterobacter cloacae Isolated from a Patient with Bloodstream Infection.

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.

Horizontal Gene Transfer of Short-Chain Dehydrogenase Coding Genes Contribute to the Biofilm Formation and Pathogenicity on Mycobacterium grossiae sp. nov. PB739T (=DSM 104744T).

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.

Dynamics of mcr-1 prevalence and mcr-1-positive Escherichia coli after the cessation of colistin use as a feed additive for animals in China: a prospective cross-sectional and whole genome sequencing-based molecular epidemiological study.

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<0·0001). A similar decrease occurred in samples from most sources in Guangzhou (959 [19%] of 5003 samples in 2016; 238 [5%] of 4489 samples in 2018; p<0·0001). The population structure of MCRPEC was diverse (23 sequence clusters); sequence type 10 clonal complex isolates were predominant (247 [36%] of 688). MCRPEC causing infection in patients admitted to hospital were genetically more distinct and appeared less affected by the ban. mcr-1 was predominantly found on plasmids (632 [92%] of 688). Common mcr-1 plasmid types included IncX4, IncI2, and IncHI2 (502 [76%] of 656); 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 single nucleotide polymorphisms, and 30 accessory genes were also significantly different after the ban (Benjamini-Hochberg-adjusted p<0·05), consistent with rapid genetic adaptation in response to changing selection pressures. INTERPRETATION: A rapid, ecosystem-wide, decline in mcr-1 was observed after the use of colistin in animal feed was banned, with associated genetic changes in MCRPEC. Withdrawal of antimicrobials from animal feed should be an important One Health measure contributing to the wider control of antimicrobial resistance globally. FUNDING: National Natural Science Foundation of China.