A Dietary Source of High Level of Fluoroquinolone Tolerance in mcr-Carrying Gram-Negative Bacteria.

The emergence of antibiotic tolerance, characterized by the prolonged survival of bacteria following antibiotic exposure, in natural bacterial populations, especially in pathogens carrying antibiotic resistance genes, has been an increasing threat to public health. However, the major causes contributing to the formation of antibiotic tolerance and underlying molecular mechanisms are yet poorly understood. Herein, we show that potassium sorbate (PS), a widely used food additive, triggers a high level of fluoroquinolone tolerance in bacteria carrying mobile colistin resistance gene mcr. Mechanistic studies demonstrate that PS treatment results in the accumulation of intracellular fumarate, which activates bacterial two-component system and decreases the expression level of outer membrane protein OmpF, thereby reducing the uptake of ciprofloxacin. In addition, the supplementation of PS inhibits aerobic respiration, reduces reactive oxygen species production and alleviates DNA damage caused by bactericidal antibiotics. Furthermore, we demonstrate that succinate, an intermediate product of the tricarboxylic acid cycle, overcomes PS-mediated ciprofloxacin tolerance. In multiple animal models, ciprofloxacin treatment displays failure outcomes in PS preadministrated animals, including comparable survival and bacterial loads with the vehicle group. Taken together, our works offer novel mechanistic insights into the development of antibiotic tolerance and uncover potential risks associated with PS use.

NK cell marker gene-based model shows good predictive ability in prognosis and response to immunotherapies in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth leading cause of malignancy worldwide, and its progression is influenced by the immune microenvironment. Natural killer (NK) cells are essential in the anti-tumor response and have been linked to immunotherapies for cancers. Therefore, it is important to unify and validate the role of NK cell-related gene signatures in HCC. In this study, we used RNA-seq analysis on HCC samples from public databases. We applied the ConsensusClusterPlus tool to construct the consensus matrix and cluster the samples based on their NK cell-related expression profile data. We employed the least absolute shrinkage and selection operator regression analysis to identify the hub genes. Additionally, we utilized the CIBERSORT and ESTIMATE web-based methods to perform immune-related evaluations. Our results showed that the NK cell-related gene-based classification divided HCC patients into three clusters. The C3 cluster was activated in immune activation signaling pathways and showed better prognosis and good clinical features. In contrast, the C1 cluster was remarkably enriched in cell cycle pathways. The stromal score, immune score, and ESTIMATE score in C3 were much higher than those in C2 and C1. Furthermore, we identified six hub genes: CDC20, HMOX1, S100A9, CFHR3, PCN1, and GZMA. The NK cell-related genes-based risk score subgroups demonstrated that a higher risk score subgroup showed poorer prognosis. In summary, our findings suggest that NK cell-related genes play an essential role in HCC prognosis prediction and have therapeutic potential in promoting NK cell antitumor immunity. The six identified hub genes may serve as useful biomarkers for novel therapeutic targets.

Circ_0073228 serves as a competitive endogenous ribonucleic acid to facilitate proliferation and inhibit apoptosis of hepatocellular carcinoma cells by the miR-139-5p/deoxyribonuclease II axis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignancy and has extremely poor prognosis and outcome. Homo sapiens deoxyribonuclease II (DNASE2) has been reported to participate in HCC progression. Here, the role of DNASE2 in HCC cells and the putative upstream circRNA that mediates DNASE2 expression was investigated. METHODS: The expression of RNAs in liver hepatocellular carcinoma (LIHC) samples was analyzed by bioinformatic analysis. The proliferation, apoptosis, migration, invasion, and gene expression in HCC cells were investigated using a Cell Counting Kit -8, colony formation, flow cytometry analysis, wound healing, transwell, western blotting, and a quantitative reverse transcriptase-PCR. The binding relationship among circ_0073228, miR-139-5p and DNASE2 was measured by RNA pulldown and luciferase reporter assays. RESULTS: DNASE2 knockdown inhibited proliferation and promoted apoptosis of HCC cells, whereas DNASE2 overexpression showed the opposite results. miR-139-5p targeted DNASE2 and suppressed its expression. Overexpression of miR-139-5p inhibited malignant phenotypes of HCC cells. RPS23-derived circ_0073228, which bound to miR-139-5p, was found to be upregulated in HCC cells. Inhibition of miR-139-5p or overexpression of DNASE2 counteracted the inhibitory effects of circ_0073228 knockdown on HCC cell progression. CONCLUSIONS: circ_0073228 serves as an oncogene to facilitate growth and inhibit apoptosis of HCC cells by regulating the miR-139-5p/DNASE2 axis.

The activity and mechanism of vidofludimus as a potent enzyme inhibitor against NDM-1-positive E. coli.

New Delhi metallo-ß-lactamase-1 (NDM-1) is the most important and prevalent enzyme among all metallo-ß-lactamases. NDM-1 can hydrolyze almost all-available ß-lactam antibiotics including carbapenems, resulting in multidrug resistance, which poses an increasing clinical threat. However, there is no NDM-1 inhibitor approved for clinical treatment. Therefore, identifying a novel and potential enzyme inhibitor against NDM-1-mediated infections is an urgent need. In this study, vidofludimus was identified as a potential NDM-1 inhibitor by structure-based virtual screening and an enzyme activity inhibition assay. Vidofludimus significantly inhibited NDM-1 hydrolysis activity with a significant dose-dependent effect. When the vidofludimus concentration was 10 µg/ml, the inhibition rate and 50% inhibitory concentration were 93.3% and 13.8 ± 0.5 µM, respectively. In vitro, vidofludimus effectively restored the antibacterial activity of meropenem against NDM-1-positive Escherichia coli (E. coli), and the minimum inhibitory concentration of meropenem was decreased from 64 µg/ml to 4 µg/ml, a 16-fold reduction. The combination of vidofludimus and meropenem showed a significant synergistic effect with a fractional inhibitory concentration index of 0.125 and almost all the NDM-1-positive E. coli were killed within 12 h. Furthermore, the synergistic therapeutic effect of vidofludimus and meropenem in vivo was evaluated in mice infected with NDM-1 positive E. coli. Compared with the control treatment, vidofludimus combined with meropenem significantly improved the survival rate of mice infected with NDM-1-positive E. coli (P < 0.05), decreased the white blood cell count, the bacterial burden and inflammatory response induced by NDM-1-positive E. coli (P < 0.05), and alleviated histopathological damage in infected mice. It was demonstrated by molecular dynamic simulation, site-directed mutagenesis and biomolecular interaction that vidofludimus could interact directly with the key amino acids (Met67, His120, His122 and His250) and Zn2+ in the active site of NDM-1, thereby competitively inhibiting the hydrolysis activity of NDM-1 on meropenem. In summary, vidofludimus holds promise as anNDM-1 inhibitor, and the combination of vidofludimus and meropenem has potential as a therapeutic strategy for NDM-1-mediated infections.

Gastrodin ameliorates atherosclerosis by inhibiting foam cells formation and inflammation through down-regulating NF-κB pathway.

BACKGROUND: Gastrodin is an effective polyphenol extracted from Chinese natural herbal Gastrodiae elata Blume, which exhibits antioxidant and anti-inflammatory effects. It has been reported to benefit neurodegenerative diseases, but the effect of Gastrodin on atherosclerosis and the underlying mechanisms remain elusive. The aim of this study is to investigate the function and mechanism of Gastrodin in atherosclerosis. METHODS: Atherosclerosis mouse model was established by fed low density lipoprotein receptor-deficient (Ldlr-/-) mice with a high fat diet (HFD, 20% fat and 0.5 cholesterol) for 8 weeks and Gastrodin was administered daily via oral gavage. Plasma lipid levels were measured using commercial kits. En face and aortic sinus lipid accumulation were analyzed with Oil Red O staining. In vitro cell models using foam cell formation model and classical atherosclerosis inflammation model, macrophages were incubated with oxygenized low-density lipoproteins (ox-LDL) or lipopolysaccharide (LPS) in the presence of different concentration of Gastrodin or vehicle solution. Foam cell formation and cellular lipid content were evaluated by Oil Red O staining and intracellular lipids extraction analysis. Gene expression and proteins related to cholesterol influx and efflux were examined by quantitative reverse transcription PCR (RT-qPCR) and western blotting analysis. Furthermore, the effect of Gastrodin on LPS induced macrophage inflammatory responses and NF-κB pathway were evaluated by RT-qPCR and western blotting analysis. RESULTS: Gastrodin administration reduced the body weight, plasma lipid levels in Ldlr-/- mice after fed a high fat diet. Oil Red O staining showed Gastrodin-treated mice displayed less atherosclerosis lesion area. Furthermore, Gastrodin treatment significantly ameliorated ox-LDL-induced macrophage-derived foam cells formation through suppressing genes expression related to cholesterol efflux including scavenger receptor class B and ATP-binding cassette transporter A1. Moreover, Gastrodin markedly suppressed pro-inflammatory cytokines secretion and LPS induced inflammatory response in macrophage through downregulating NF-κB pathway. CONCLUSIONS: Our study demonstrated that Gastrodin attenuates atherosclerosis by suppressing foam cells formation and LPS-induced inflammatory response and represents a novel therapeutic target for the treatment of atherosclerosis.

Identification and Characterization of an Ageing-Associated 13-lncRNA Signature That Predicts Prognosis and Immunotherapy in Hepatocellular Carcinoma.

Background: In cancer pathology, cell senescence not only alters cell function but also reshapes the immune microenvironments in tumours. However, the association between cell senescence, tumour microenvironment, and disease progression of hepatocellular carcinoma (HCC) is yet to be fully understood. Therefore, the role of cell senescence-related genes and long noncoding RNAs (lncRNAs) in evaluating the clinical prognosis and immune cell infiltration (ICI) of HCC patients requires further investigation. Methods: The limma R package was utilised to investigate differentially expressed genes according to the multiomics data. The CIBERSORT R package was utilised to assess ICI, and unsupervised cluster analysis was conducted using the R software's ConsensusClusterPlus package. A polygenic prognostic model of lncRNAs was constructed by conducting univariate and least absolute shrinkage and selection operator (Lasso) cox proportional-hazards regression analyses. The time-dependent receiver operating characteristic (ROC) curves were used for validation. We utilised the survminer R package to evaluate the tumour mutational burden (TMB). Moreover, the gene set enrichment analysis (GSEA) helped in pathway enrichment analysis, and the immune infiltration level of the model was evaluated using the IMvigor210 cohort. Results: The identification of 36 prognosis-related genes was achieved based on their differential expression between healthy and liver cancer tissues. Liver cancer individuals were categorised into 3 independent senescence subtypes using the gene list, revealing considerable survival differences (variations). We observed that the prognosis of patients in the ARG-ST2 subtype was substantially better as compared to that in the ARG-ST3 subtype. Differences were observed in gene expression profiles among the three subtypes, with the differentially expressed genes predominantly associated with cell cycle control. The enrichment of upregulated genes in the ARG-ST3 subtype was observed in pathways related to biological processes, for instance, organelle fission, nuclear division, and chromosome recombination. ICI in the ARG-ST1 and ARG-ST2 subtypes, with relatively better prognosis, was substantially higher as compared to the ARG-ST3 subtype. Furthermore, a risk-score model, which can be employed as a reliable prognostic factor in an independent manner for individuals suffering from liver cancer, was constructed based on 13 cell senescence-related lncRNAs (MIR99AHG, LINC01224, LINC01138, SLC25A30AS1, AC006369.2, SOCS2AS1, LINC01063, AC006037.2, USP2AS1, FGF14AS2, LINC01116, KIF25AS1, and AC002511.2). The individuals with higher risk scores had noticeably poor prognoses in contrast with those having low-risk scores. Moreover, increased levels of TMB and ICI were observed in individuals with low-risk scores and gaining more benefit from immune checkpoint therapy. Conclusion: Cell senescence is an essential factor in HCC onset and progression. We identified 13 senescence-related lncRNAs as HCC prognostic markers, which can help understand their function in the onset and progression of HCC and guide clinical diagnosis and treatment.

A review of literature: role of long noncoding RNA TPT1-AS1 in human diseases

Human diseases are multifactorial processes mainly driven by the intricate interactions of genetic and environmental factors. Long noncoding RNAs (lncRNAs) represent a type of non-coding RNAs with more than 200 nucleotides. Multiple studies have demonstrated that the dysregulation of lncRNAs is associated with complex biological as well as pathological processes through various mechanism, especially the regulation of gene transcription and related signal transduction pathways. Moreover, an increasing number of studies have explored lncRNA-based clinical applications in different diseases. For instance, the lncRNA Tumor Protein Translationally Controlled 1 (TPT1) Antisense RNA 1 (TPT1-AS1) was found to be dysregulated in several types of disease and strongly associated with patient prognosis and diverse clinical features. Recent studies have also documented that TPT1-AS1 modulates numerous biological processes through multiple mechanisms, including cell proliferation, apoptosis, autophagy, invasion, migration, radiosensitivity, chemosensitivity, stemness, and extracellular matrix (ECM) synthesis. Furthermore, TPT1-AS1 was regarded as a promising biomarker for the diagnosis, prognosis and treatment of several human diseases. In this review, we summarize the role of TPT1-AS1 in human diseases with the aspects of its expression, relevant clinical characteristics, molecular mechanisms, biological functions, and subsequent clinical applications (AU)

Nicotinamide Mononucleotide Ameliorates Sleep Deprivation-Induced Gut Microbiota Dysbiosis and Restores Colonization Resistance against Intestinal Infections.

Gut microbiota-mediated colonization resistance (CR) is crucial in protecting the host from intestinal infections. Sleep deprivation (SD) is an important contributor in the disturbances of intestinal homeostasis. However, whether and how SD affects host CR remains largely unknown. Here, it is shown that SD impairs intestinal CR in mice, whereas nicotinamide mononucleotide (NMN) supplementation restores it. Microbial diversity and metabolomic analyses suggest that gut microbiota and metabolite profiles in SD-treated mice are highly shaped, whereas NMN reprograms these differences. Specifically, the altered gut microbiota in SD mice further incurs the disorder of secondary bile acids pool accompanied by a decrease in deoxycholic acid (DCA). Conversely, NMN supplementation retakes the potential benefits of DCA, which is associated with specific gut microbiota involved in primary bile acids metabolic flux. In animal models of infection, DCA is effective in preventing and treating bacterial infections when used alone or in combination with antibiotics. Mechanistically, DCA alone disrupts membrane permeability and aggravates oxidative damage, thereby reducing intestinal pathogen burden. Meanwhile, exogenous DCA promotes antibiotic accumulation and destroys oxidant-antioxidant system, thus potentiating antibiotic efficacy. Overall, this work highlights the important roles of gut microbiota and bile acid metabolism in the maintenance of intestinal CR.

A review of literature: role of long noncoding RNA TPT1-AS1 in human diseases.

Human diseases are multifactorial processes mainly driven by the intricate interactions of genetic and environmental factors. Long noncoding RNAs (lncRNAs) represent a type of non-coding RNAs with more than 200 nucleotides. Multiple studies have demonstrated that the dysregulation of lncRNAs is associated with complex biological as well as pathological processes through various mechanism, especially the regulation of gene transcription and related signal transduction pathways. Moreover, an increasing number of studies have explored lncRNA-based clinical applications in different diseases. For instance, the lncRNA Tumor Protein Translationally Controlled 1 (TPT1) Antisense RNA 1 (TPT1-AS1) was found to be dysregulated in several types of disease and strongly associated with patient prognosis and diverse clinical features. Recent studies have also documented that TPT1-AS1 modulates numerous biological processes through multiple mechanisms, including cell proliferation, apoptosis, autophagy, invasion, migration, radiosensitivity, chemosensitivity, stemness, and extracellular matrix (ECM) synthesis. Furthermore, TPT1-AS1 was regarded as a promising biomarker for the diagnosis, prognosis and treatment of several human diseases. In this review, we summarize the role of TPT1-AS1 in human diseases with the aspects of its expression, relevant clinical characteristics, molecular mechanisms, biological functions, and subsequent clinical applications.

Clinical application of traditional Chinese medicine powder in the treatment of acute and chronic wounds.

This study aimed to explore the clinical application and efficacy of traditional Chinese medicine (TCM) powder in the treatment of acute and chronic wounds. Eighty patients with a wound infection were randomly and equally divided into a control group and an observation group. Gauze padding containing furacilin was used to dress the infected wounds of the control group. TCM powder was used to treat the wounds of the observation group. The total response rate of the observation group was significantly higher than the control group (P = .017). The colour and exudate volume scores in the observation group were lower than the control group, and the differences between the two groups were statistically significant (P < .05). The time to the appearance of new epithelium and time to the wound healing of the burns in the observation group were shorter than the control group, and the differences were statistically significant (P < .05). The TCM powder absorbed a large amount of necrotic tissue and exudate from the wound surface, cleared heat and toxins, and activated blood circulation. It also resolved blood stasis, eliminated pus, and allowed for new skin growth, as well as regenerating muscle.

Molecular Epidemiology and Colistin-Resistant Mechanism of mcr-Positive and mcr-Negative Escherichia coli Isolated From Animal in Sichuan Province, China.

Colistin is the last line of defense for the treatment of multidrug-resistant gram-negative bacterial infections. However, colistin resistance is gradually increasing worldwide, with resistance commonly regulated by two-component system and mcr gene. Thus, this study aimed to investigate molecular epidemiology and colistin-resistant mechanism of mcr-positive and mcr-negative Escherichia coli isolates from animal in Sichuan Province, China. In this study, a total of 101 colistin-resistant E. coli strains were isolated from 300 fecal samples in six farms in Sichuan Province. PCR was used to detect mcr gene (mcr-1 to mcr-9). The prevalence of mcr-1 in colistin-resistant E. coli was 53.47% (54/101), and the prevalence of mcr-3 in colistin-resistant E. coli was 10.89% (11/101). The colistin-resistant E. coli and mcr-1-positive E. coli showed extensive antimicrobial resistance profiles. For follow-up experiments, we used 30 mcr-negative and 30 mcr-1-positive colistin-resistant E. coli isolates and E. coli K-12 MG1655 model strain. Multi-locus sequence typing (MLST) of 30 strains carrying mcr-1 as detected by PCR identified revealed six strains (20%) of ST10 and three strains (10%) of each ST206, ST48, and ST155 and either two (for ST542 and 2539) or just one for all other types. The conjugation experiment and plasmid replicon type analysis suggest that mcr-1 was more likely to be horizontally transferred and primarily localized on IncX4-type and IncI2-type plasmid. The ST diversity of the mcr-1 indicated a scattered and non-clonal spreading in mcr-1-positive E. coli. Twenty-eight mcr-negative colistin-resistant E. coli isolates carried diverse amino acid alterations in PmrA, PmrB, PhoP, PhoQ, and MgrB, whereas no mutation was found in the remaining isolates. The finding showed the high prevalence of colistin resistance in livestock farm environments in Sichuan Province, China. Our study demonstrates that colistin resistance is related to chromosomal point mutations including the two-component systems PhoP/PhoQ, PmrA/PmrB, and their regulators MgrB. These point mutations may confer colistin resistance in mcr-negative E. coli. These findings help in gaining insight of chromosomal-encoded colistin resistance in E. coli.

Prevalence and Characteristic of Swine-Origin mcr-1-Positive Escherichia coli in Northeastern China.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 is threatening the last-line role of colistin in human medicine. With mcr-1-positive Escherichia coli (E. coli) isolated from food animal being frequently reported in China, the prevalence of mcr-1 in food animal has attracted public attention. In the present study, a total of 105 colistin-resistant E. coli strains were isolated from 200 fecal samples collected from six swine farms in northeastern China. mcr-PCR revealed that the prevalence of mcr-1 in colistin-resistant E. coli was 53.33% (56/105). mcr-1-positive E. coli showed extensive antimicrobial resistance profiles with the presence of additional resistance genes, increased expression of multidrug efflux pump-associated genes, and increased biofilm formation ability. MLST differentiated all the mcr-1-positive E. coli into 25 sequence types (STs) and five unknown ST, and the most common ST was ST10 (n = 11). By phylogenetic group classification, the distribution of all mcr-1-positive E. coli belonging to groups A, B1, B2, and D was 46.43, 35.71, 5.36, and 5.36%, respectively. Conjugation experiment demonstrated that most of the mcr-1 were transferable at frequencies of 2.68 × 10-6-3.73 × 10-3 among 30 representative mcr-1-positive E. coli. The plasmid replicon types IncI2 (n = 9), IncX4 (n = 5), IncHI2 (n = 3), IncN (n = 3), and IncP (n = 1) were detected in the transconjugants. The results of growth assay, competition experiment, and plasmid stability testing showed that acquisition of mcr-1-harboring plasmids could reduce the fitness of bacterial hosts, but mcr-1 remained stable in the recipient strain. Due to the potential possibility of these mcr-1-positive E. coli being transmitted to humans through the food chain or through horizontal transmission, therefore, it is necessary to continuously monitor the prevalence and dissemination of mcr-1 in food animal, particularly in swine.

Resistance Detection and Transmission Risk Analysis of Pig-Derived Pathogenic Escherichia coli in East China.

Objective: Antibiotics play an essential role in the treatment and prevention of diseases in pig farms. However, the irrational use of antibiotics leads to the emergence of multi-drug resistance of bacteria, which poses a critical threat to the efficacy of antibiotic treatments. Therefore, the study is designed to analyze the drug resistance of pathogenic Escherichia coli isolated from large-scale pig farms in East China, which provides a theoretical basis for precisely targeted clinical drugs in swine farms. Method: The pathogenic E. coli were isolated and identified from clinical samples of swine farms, and the drug resistance of pathogenic E. coli was detected by antimicrobial susceptibility test (AST) and minimum inhibitory concentration test (MIC). Moreover, the prevalence of plasmid-mediated ß-lactam resistance genes was analyzed by PCR. Results: A total of 67 pathogenic E. coli were isolated from 152 samples collected from 20 large-scale pig farms in East China. All isolated pathogenic E. coli are associated with severe drug resistance. Moreover, 70% of isolated pathogenic E. coli is resistant to more than four antibiotics. Besides, there were 19 serotypes including O2, O4, O5, O6, O14, O26, O38, O42, O49, O57, O92, O93, O95, O101, O121, O131, O143, O158, and O161, of which the O4 and O92 serotype were the main serotypes in swine farms. The main extended-spectrum beta-lactamases (ESBLs)-encoding genes in East China were bla CTX-M, bla TEM, and bla OXA by the detection of the ESBLs encoding genes of porcine pathogenic E. coli. The conjugation assays showed that a total of 30 transconjugants were obtained by conjugation, which indicated that drug resistance genes could be transmitted horizontally through conjugative plasmids. Conclusion: The isolated pathogenic E. coli were all multi-drug resistant, and especially O4 and O92 were the main serotypes. The ß-lactam resistance genes were prevalent in large-scale pig farms in East China, which provided a theoretical basis for the prevention and control of pig-derived pathogenic E. coli in the future.

Wild-type cutoff for Apramycin against Escherichia coli.

BACKGROUND: Apramycin is used exclusively for the treatment of Escherichia coli (E.coli) infections in swine around the world since the early 1980s. Recently, many research papers have demonstrated that apramycin has significant in vitro activity against multidrug-resistant E.coli isolated in hospitals. Therefore, ensuring the proper use of apramycin in veterinary clinics is of great significance of public health. The objectives of this study were to develop a wild-type cutoff for apramycin against E.coli using a statistical method recommended by Clinical and Laboratory Standards Institute (CLSI) and to investigate the prevalence of resistance genes that confer resistance to apramycin in E. coli. RESULTS: Apramycin susceptibility testing of 1230 E.coli clinical isolates from swine were determinded by broth microdilution testing according to the CLSI document M07-A9. A total number of 310 E.coli strains from different minimum inhibitory concentration (MIC) subsets (0.5-256 µg/mL) were selected for the detection of resistance genes (aac(3)-IV; npmA; apmA) in E. coli by PCR. The percentage of E. coli isolates at each MIC (0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/mL) was 0.08, 0.08, 0.16, 2.93, 31.14, 38.86, 12.85, 2.03, 1.46, and 10.41%. The MIC50 and MIC90 were 16 and 64 µg/mL. All the 310 E.coli isolates were negative for npmA and apmA gene, and only the aac(3)-IV gene was detected in this study. CONCLUSIONS: The wild-type cutoff for apramycin against E.coli was defined as 32 µg/mL. The prevelance of aac(3)-IV gene mainly concentrated in these MIC subsets 'MIC ≥ 64 µg/ mL', which indicates that the wild-type cutoff established in our study is reliable. The wild-type cutoff offers interpretion criteria of apramycin susceptibility testing of E.coli.

Antimicrobial Resistance and Virulence Profiles of mcr-1-Positive Escherichia coli Isolated from Swine Farms in Heilongjiang Province of China.

ABSTRACT: The emergence and global distribution of the mcr-1 gene for colistin resistance have become a public concern because of threats to the role of colistin as the last line of defense against some bacteria. Because of the prevalence of mcr-1-positive Escherichia coli isolates in food animals, production of these animals has been regarded as one of the major sources of amplification and spread of mcr-1. In this study, 249 E. coli isolates were recovered from 300 fecal samples collected from swine farms in Heilongjiang Province, People's Republic of China. Susceptibility testing revealed that 186 (74.70%) of these isolates were colistin resistant, and 86 were positive for mcr-1. The mcr-1-positive isolates had extensive antimicrobial resistance profiles and additional resistance genes, including blaTEM, blaCTX-M, aac3-IV, tet(A), floR, sul1, sul2, sul3, and oqxAB. No mutations in genes pmrAB and mgrB were associated with colistin resistance. Phylogenetic group analysis revealed that the mcr-1-positive E. coli isolates belonged to groups A (52.33% of isolates), B1 (33.72%), B2 (5.81%), and D (8.14%). The prevalence of the virulence-associated genes iutA, iroN, fimH, vat, ompA, and traT was moderate. Seven mcr-1-positive isolates were identified as extraintestinal pathogenic. Among 20 mcr-1-positive E. coli isolates, multilocus sequence typing revealed that sequence type 10 was the most common (five isolates). The conjugation assays revealed that the majority of mcr-1 genes were transferable at frequencies of 7.05 × 10-7 to 7.57 × 10-4. The results of this study indicate the need for monitoring and minimizing the further dissemination of mcr-1 among E. coli isolates in food animals, particularly swine.

The prevalence and mechanism of fluoroquinolone resistance in Escherichia coli isolated from swine farms in China.

BACKGROUND: It has been demonstrated that swine waste is an important reservoir for resistant genes. Moreover, the bacteria carrying resistant genes and originating from swine feces and wastewater could spread to the external environment. Fluoroquinolones (FQs) are widely used in livestock and poultry for the treatment of bacterial infection. However, resistance to FQs has increased markedly. RESULTS: In this study, swine feces and wastewater were sampled from 21 swine farms of seven provinces in China to investigate the prevalence of FQ resistance, including plasmid-mediated fluoroquinolone resistance (PMQR) genes and the occurrence of target mutations. All isolates showed moderate rate of resistance to norfloxacin (43.0%), ciprofloxacin (47.6%), ofloxacin (47.0%) and levofloxacin (38.8%). The percentage of strains resistant to the four FQs antimicrobials was positively correlated with the danofloxacin (DANO) MIC. Among the 74 FQ-resistant isolates, 39 (52.70%) had mutations in gyrA (S83L and D87 to N, Y, G, or H), 21 (28.38%) had mutations in parC (S80I and E84K), 2 (2.70%) had mutations in parE (I355T and L416F), 26 (35.14%) had mutations in marR (D67N and G103S), 1 (1.35%) had mutations in acrR (V29G). While, no mutation was found in gyrB. There were 7 (9.46%) strains carried the qnrS gene, 29 (39.19%) strains carried the oqxAB gene, and 9 (12.16%) strains carried the aac (6')-Ib-cr gene. In addition, the conjugation assays showed that qnrS, oqxAB and aac (6')-Ib-cr could be successfully transferred to E. coli J53 from 4 (57.1%), 20 (69.0%) and 5 (55.6%) donor strains, respectively. There were no qnrA, qnrB, qnrC, qnrD and qepA genes detected. CONCLUSION: The present study showed that DANO-resistant E. coli strains isolated from swine farms had significant cross-resistance to other four FQs antimicrobials. Further study revealed that the resistance mechanisms of swine-derived E. coli to FQs may be attributable to the occurrence of chromosomal mutations (gyrA, parC, parE, marR and acrR genes double-site or single-site mutation) and the presence of PMQR genes (qnrS, oqxAB and aac (6')-Ib-cr). To the best of our knowledge, one novel mutation marR-D67N was found to be associated with FQ resistance, two mutations parE-L416F and acrR-V29G have never been reported in China.

Construction and evaluation of a prognosis lncRNA model for hepatocellular carcinoma.

Current studies indicate that long non-coding RNA (lncRNA) is often abnormally expressed in hepatocellular carcinoma (HCC). We intend to generate a multi-lncRNA signal to improve the prognosis of HCC. By analyzing 12 pairs of HCC and adjacent normal mucosal tissues, 3900 differentially expressed lncrnas were identified as candidate biomarkers for the prognosis of HCC. Then, the 12-lncrna signature was constructed using the LASSO Cox regression method and verified in the TCGA training dataset. Finally, we established a novel 12-lncrna signature that was significantly associated with overall survival (OS) in the training data set. With the use of 12-lncrna markers, patients in the training cohort were divided into high-risk and low-risk groups with significant OV differences (P < .0001). Similar results were consistent in the TCGA verification dataset (P = .046). Multivariate Cox model was used to analyze and construct the risk scores of selected key lncRNA and AJCC stages. The results showed that, compared with AJCC stages, lncRNA-based risk scores were another important factor affecting the OS of patients. We found that risk scores based on lncRNA have a stronger prediction ability than the AJCC stage alone on 4-year OS. For 4-year survival rates, prediction combined with the lncRNA risk score and AJCC stage, model effectiveness (sensitivity and specificity) has reached to 0.750. To further explore the biological processes involved in prognostic lncRNA, all HCC samples in TCGA are divided into two groups according to the median lncRNA risk score, and analyzed the gene enrichment of high expression genes and low expression genes in KEGG data using goana in limma. The results suggest that the genes associated with tumor pathways, such as PI3K-Akt and ECM-receptor interaction, are highly expressed in the high risk group.

Prevalence and molecular epidemiology characteristics of carbapenem-resistant Escherichia coli in Heilongjiang Province, China.

OBJECTIVE: This retrospective study was conducted to determine the prevalence and molecular epidemiology characteristics of carbapenem-resistant Escherichia coli (CRE). METHODS: A total of 593 Escherichia coli (E. coli) isolates were recovered from pigs and urban river from 2009 to 2014 in Heilongjiang Province of China. Forty CRE including 22 strains isolated from fecal samples of pigs and 18 strains isolated from water samples were selected. PCR detection of resistance determinants, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and phylogenetic groups were performed to characterize CRE isolates. Conjugation experiments, plasmid stability testing, PCR-based replicon typing (PBRT), and PCR mapping were conducted to analyze bla NDM-carrying plasmids. In vitro time-growth studies and competition experiments were carried out to assess the fitness impact of NDM carriage. RESULTS: Five NDM-1-positive E. coli isolates were identified from water samples. Genetic environment analysis revealed that a cluster of genes (ISAba125-bla NDM-1-ble MBL-ΔtrpF) was detected in all of the NDM-1-positive isolates. Conjugation assays showed that bla NDM-1 could be successfully transferred to E. coli J53 from 5 donor strains at frequencies of 4.6×10-5 to 2.6×10-2. The plasmids from all transconjugants belonged to different plasmid replicon types including IncA/C (n=2), IncFII (n=1) and IncX3 (n=2). In vitro time-growth studies revealed that bla NDM-1 did not have a significant impact on cell proliferation. Meanwhile, competition experiments showed that the acquisition of bla NDM-1 can place an energy burden on the bacterial host and incur fitness cost. However, plasmid stability testing showed that bla NDM-1-carrying plasmid remained stable in the hosts after seven passages without antimicrobial selection. CONCLUSION: The study revealed the early molecular epidemiology and dissemination characteristics of CRE. In addition, the overall antimicrobial resistance in E. coli recovered from water samples is higher than the strains isolated from fecal samples of pigs. Furthermore, we isolated and identified five NDM-1-producing E. coli strains from water samples.

NH4Cl affects the expression of Wnt/ß-catenin pathway in hepatocytes.

We intended to explore whether NH4Cl influences the viability and regulates the expression of Wnt/ß-catenin pathway in hepatocytes. The Chang liver cell line was used and cultured with different concentrations of NH4Cl (2.5, 5, 10, 20, 40, and 50 mmol/L) for 12, 24, and 48 h. The viability of hepatocytes was detected by MTT assay. The mRNA and protein expression level was analyzed with qRT-PCR and Western blotting, respectively. NH4Cl concentration significantly affects the viability of hepatocytes. With the increase of NH4Cl concentration, the viability of hepatocytes was decreased, accordingly. The mRNA and protein expression of Wnt1, ß-catenin, and cyclin D was significantly increased after treatment with low concentrations of NH4Cl as compared with the control group, whereas their expression levels were decreased after treatment with high concentrations of NH4Cl. The mRNA and protein expression of Wnt1, ß-catenin, and cyclin D was also significantly increased after treatment with NH4Cl for a short period as compared with the control group, whereas their expression levels were decreased after treatment with NH4Cl for a long period. In addition, we found NH4Cl treatment significantly reversed the results after RNA silencing of Wnt1 in hepatocytes. NH4Cl influences the viability of hepatocytes and affects the expression of Wnt/ß-catenin pathway in hepatocytes.