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 ...
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 -Δ trpF ) 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.
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.
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.
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.
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
The emergence and dissemination of mcr-1 have become a public concern worldwide. And food animal production has been singled out as responsible for amplification and spread of mcr-1 . Herein, a total of 249 porcine Escherichia coli ( E. coli ) were isolated from the 300 fecal samples in Heilongjiang province of China. Susceptibility testing revealed 186 (74.70%) E. coli were colistin-resistant. And a total of 86 mcr-1 -positive E. coli were detected. The mcr-1 -positive E. coli showed extensive antimicrobial resistance profiles with the presence of additional resistance genes, including TEM1 , CTX-M , aac3-IV , tetA , flor , sul1 , sul2 , sul3 , oqxAB . No mutations in pmrA, pmrB and mgrB were found to be associated with colistin resistance. By phylogenetic group classification, the distribution of all mcr-1 -positive E. coli belonging to group A, B1, B2 and D was 52.33%, 33.72%, 5.81%, 8.14%, respectively. The presence of virulence-associated genes iutA, iron, fimH, vat, ompA and traT was at moderate frequencies. And 7 mcr -positive E. coli belonged to ExPEC. Among 20 representative mcr-1 -positive E. coli, MLST showed that the most common ST was ST 10 (n=5). The conjugation assays showed that the majority of mcr-1 were transferable at frequencies of 7.05×10 −7 to 7.57×10 −4 . Therefore, the results of this study revealed the necessity of monitoring and minimizing the further dissemination of mcr-1 in food animals, particular in swine.
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.
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