Unveiling the genetic architecture and transmission dynamics of a novel multidrug-resistant plasmid harboring blaNDM-5 in E. Coli ST167: implications for antibiotic resistance management.

BACKGROUND: The emergence of multidrug-resistant (MDR) Escherichia coli strains poses significant challenges in clinical settings, particularly when these strains harbor New Delhi metallo-ß-lactamase (NDM) gene, which confer resistance to carbapenems, a critical class of last-resort antibiotics. This study investigates the genetic characteristics and implications of a novel blaNDM-5-carrying plasmid pNDM-5-0083 isolated from an E. coli strain GZ04-0083 from clinical specimen in Zhongshan, China. RESULTS: Phenotypic and genotypic evaluations confirmed that the E. coli ST167 strain GZ04-0083 is a multidrug-resistant organism, showing resistance to diverse classes of antibiotics including ß-lactams, carbapenems, fluoroquinolones, aminoglycosides, and sulfonamides, while maintaining susceptibility to monobactams. Investigations involving S1 pulsed-field gel electrophoresis, Southern blot analysis, and conjugation experiments, alongside genomic sequencing, confirmed the presence of the blaNDM-5 gene within a 146-kb IncFIB plasmid pNDM-5-0083. This evidence underscores a significant risk for the horizontal transfer of resistance genes among bacterial populations. Detailed annotations of genetic elements-such as resistance genes, transposons, and insertion sequences-and comparative BLAST analyses with other blaNDM-5-carrying plasmids, revealed a unique architectural configuration in the pNDM-5-0083. The MDR region of this plasmid shares a conserved gene arrangement (repA-IS15DIV-blaNDM-5-bleMBL-IS91-suI2-aadA2-dfrA12) with three previously reported plasmids, indicating a potential for dynamic genetic recombination and evolution within the MDR region. Additionally, the integration of virulence factors, including the iro and sit gene clusters and enolase, into its genetic architecture poses further therapeutic challenges by enhancing the strain's pathogenicity through improved host tissue colonization, immune evasion, and increased infection severity. CONCLUSIONS: The detailed identification and characterization of pNDM-5-0083 enhance our understanding of the mechanisms facilitating the spread of carbapenem resistance. This study illuminates the intricate interplay among various genetic elements within the novel blaNDM-5-carrying plasmid, which are crucial for the stability and mobility of resistance genes across bacterial populations. These insights highlight the urgent need for ongoing surveillance and the development of effective strategies to curb the proliferation of antibiotic resistance.

Unidirectional alteration of methylation and hydroxymethylation at the promoters and differential gene expression in oral squamous cell carcinoma.

Background: Oral squamous cell carcinoma (OSCC) is one of the most common types of cancer worldwide. Although overall losses of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been previously observed, a genome-wide, single-base-resolution, and simultaneous mapping of 5mC and 5hmC in OSCC is still unaccomplished. Similarly, the mechanism of how 5mC and 5hmC collectively lead to abnormal gene expression in OSCC is largely unexplored. Using parallel whole-genome bisulfite sequencing (WGBS) and whole-genome oxidative bisulfite sequencing (oxWGBS), we characterized 5mC- and 5hmC-profiles at single-nucleotide resolution in paired primary OSCC samples and their normal adjacent tissues (NATs). We also analyzed the effect of 5mC- and 5hmC-modifications on differential gene expression in OSCC using multi-omics analysis. Results: An overall reduction of both 5mC and 5hmC in various genomic regions have been observed in OSCC samples. At promoter regions, a total of 6,921 differentially methylated regions and 1,024 differentially hydroxymethylated regions were identified in OSCC. Interestingly, compared to bidirectional modification with 5mC and 5hmC, unidirectional modification with 5mC and 5hmC at the promoters is associated with bigger change in the gene expression. Additionally, genes bearing unidirectional modification with 5mC and 5hmC at the promoters are enriched in signaling pathways like cell proliferation, cell differentiation, and receptor tyrosine kinase pathway that are essential for the tumorigenesis. Finally, the grouped expression signature of top 20 genes bearing promoter-unidirectional-modification with 5mC and 5hmC tends to correlate with the clinical outcome of certain subtypes of head and neck squamous cell carcinoma. Conclusion: Using parallel WGBS and oxWGBS analyses, we observed an overall reduction of 5mC- and 5hmC-modifications at various genomic regions in OSCC. Unidirectional modification with 5mC and 5hmC at the promoters is associated with enhanced changes in gene expression in OSCC tissues. Furthermore, such differentially expressed genes bearing unidirectional modifications with 5mC and 5hmC at the promoters might have clinical relevance to the outcome of OSCC.

Study on the hepatotoxicity and potential mechanism of gefitinib based on CYP450 in mice and AML12 cells.

OBJECTIVES: Gefitinib is mainly used for the treatment of non-small-cell lung cancer. Hepatotoxicity is one of the main side effects of gefitinib, and seriously affects the treatment process of the disease. However, the hepatotoxicity mechanism of gefitinib remains unclear. METHODS: The hepatotoxicity of different doses of gefitinib was investigated in mice and AML-12 cells, and the possible correlation of hepatotoxicity with CYP450 was analysed. KEY FINDINGS: The toxic effects of gefitinib were confirmed by the increased liver index, decreased body weight and survival rate, injured liver function and histopathology followed 16 days of oral administration. Gefitinib (400 mg/kg) upregulated the hepatic mRNA expression of CYP1A1 and downregulated the CYP2D9 and CYP2D10 in mice. Furthermore, we verified that gefitinib produced cytotoxicity on AML-12 cells in a dose and time-dependent manner, and confirmed that gefitinib (20 µM) induced cell apoptosis, upregulated mRNA expression of CYP1A1 and downregulated CYP2D9 and CYP2D10. Pearson correlation analysis also showed that the hepatotoxicity of gefitinib was positively correlated with CYP1A1 and negatively correlated with CYP2D9 and CYP2D10. CONCLUSIONS: Our results suggested that the hepatotoxicity gefitinib may be associated with CYP1A1, CYP2D9 and CYP2D10. These findings will contribute to a better understanding of the mechanism of gefitinib hepatotoxicity.

Influence of LncRNA NKILA on Bloodstream Infection of Hypervirulent Klebsiella pneumoniae and Its Ability to Induce Delayed Neutrophil Apoptosis.

OBJECTIVE: Pneumonia due to hypervirulent Klebsiella pneumoniae (hvKP) is a high-risk subtype of pneumonia with high mortality and disability rates. An in-depth understanding of hvKP's pathogenic process and mechanism of action is the focus of achieving early disease diagnosis and early symptomatic treatment. This study conducted a preliminary analysis on the influence of lncRNA NKILA (NKILA) on hvKP, aiming at providing a new approach to the diagnosis and treatment of hvKP and laying a reliable foundation for subsequent NKILA-related studies. METHODS: Selected from our hospital from October 2016 to February 2018, 67 patients who were examined for the pathogenic microorganisms of alveolar lavage fluid were selected as the research subjects. Among them, 29 were diagnosed as hvKP (research group), and the other 38 had no pathogenic bacteria (control group). Serum and bronchoalveolar lavage fluid (BALF) NKILA and inflammatory factors were detected, and the clinical significance of NKILA was analyzed. In addition, neutrophils from research group were extracted and NKILA expression was increased to observe the alterations in cell apoptosis, respiratory burst intensity, and NF-kappa B inhibitor alpha (NF-κB) p65 protein. RESULTS: Serum and BALF levels of NKILA and inflammatory factors were higher in research group than in control group, and NKILA decreased in both cohorts after treatment (P < 0.05). NKILA had an excellent predictive effect on the occurrence of hvKP (P < 0.001) and was positively correlated with inflammatory factors (P < 0.05). Prognostic follow-up revealed that NKILA also had a good predictive value for death in hvKP patients (P < 0.05), and increased posttreatment levels predicted an increased risk of death (P < 0.05). In vitro, increased NKILA hindered the delayed apoptosis rate, decreased respiratory burst intensity of hvKP neutrophils, and activated NF-κB p65 protein (P < 0.05). CONCLUSION: With an elevated expression profile in hvKP, NKILA can induce the delayed apoptosis of neutrophils, enhance the ability of releasing inflammatory mediators, and promote the progression of hvKP via activating NF-κB p65.

Prevalence, Antimicrobial Resistance, and Relatedness of Salmonella Isolated from Chickens and Pigs on Farms, Abattoirs, and Markets in Sichuan Province, China.

This study aims at investigating the distribution, antimicrobial resistance, and genetic relationship of Salmonella isolated from 18 farms, their downstream abattoirs, and markets of chickens and pigs in Sichuan province, China. A total of 193 Salmonella isolates were identified from 693 samples with an isolation rate of 26.27% (88/335) in chickens and 29.33% (105/358) in pigs. Salmonella was isolated more frequently in abattoirs and markets than from farms. Serotypes were determined according to the White-Kauffmann-Le Minor scheme and 16 different serotypes were identified, with Derby being the most common, followed by Typhimurium and Meleagridis. Antimicrobial resistance phenotypes and genotypes were studied by using the disk diffusion method and polymerase chain reaction (PCR) amplification, respectively. Overall, 44.04% (n = 85) of all isolates were multidrug resistant (MDR) and resistance to nalidixic acid (51.30%) was the most frequently observed. blaCTX-M-55 was the most prevalent extended-spectrum ß-lactamases gene, and polymyxin resistance gene mcr-1 was present in strains with various serotypes. Multilocus sequence typing indicated that sequence type (ST) had a close relationship with serotype, and 34.20% of all strains were ST40, which was the most prevalent. The unweighted pair group method with arithmetic means (UPGMA) dendrogram of pulsed-field gel electrophoresis showed that Salmonella isolates belonging to the same serovar from different parts of the production chain were highly genetic related, indicating that Salmonella as well as resistance genes could potentially be transmitted from farms to markets. Our study highlights the fact that Salmonella isolates from chicken and pig production chain were frequently exhibiting MDR profiles, and the dissemination of MDR Salmonella from farm to market could pose significant threats to food safety and public health.

A novel type 1/2 hybrid IncC plasmid carrying fifteen antimicrobial resistance genes recovered from Proteus mirabilis in China.

IncC plasmids are of great concern as vehicles of broad-spectrum cephalosporins and carbapenems resistance genes blaCMY and blaNDM. The aim of this study was to sequence and characterize a multidrug resistance (MDR) IncC plasmid (pPm14C18) recovered from Proteus mirabilis. pPm14C18 was identified in a CMY-2-producing P. mirabilis isolate from chicken in China in 2014, and could be transferred to Escherichia coli conferring an MDR phenotype. Whole genome sequencing confirmed pPm14C18 was a novel type 1/2 hybrid IncC plasmid 165,992bp in size, containing fifteen antimicrobial resistance genes. It harboured a novel MDR mosaic region comprised of a hybrid Tn21tnp-pDUmer, in which blaCTX-M-65, dfrA32 and ereA were firstly reported in IncC plasmid. Phylogenetic relationship reconstruction based on the nucleotide sequences of the 52 IncC backbones showed all type 1 IncC plasmids were clustered into one clade, and then merged with pPm14C18 and finally with the type 2 IncC plasmids and another type 1/2 hybrid IncC plasmid pYR1. The MDR IncC plasmids in P. mirabilis of animal origin might threaten public health, which should be drawn more attention.

Prevalence of Plasmid-Mediated Fosfomycin Resistance Gene fosA3 Among CTX-M-Producing Escherichia coli Isolates from Chickens in China.

The aim of this study was to investigate the prevalence of fosfomycin resistance gene fosA3 and characterize plasmids harboring fosA3 among CTX-M-producing Escherichia coli from chickens in China. A total of 234 CTX-M-producing E. coli isolates collected from chickens from 2014 to 2016 were screened for the presence of plasmid-mediated fosfomycin resistance genes (fosA, fosA3, and fosC2). Clonal relatedness of fosA3-positive isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic environment of fosA3 was analyzed by polymerase chain reaction (PCR) mapping. Plasmids were studied by using conjugation experiments, PCR-based replicon typing and plasmid MLST. Sixty-four (27.4%) fosA3-positive E. coli isolates were identified in this study. The gene blaCTX-M-55 (31/64) was predominant among these strains, followed by blaCTX-M-14 (18/64) and blaCTX-M-65 (14/64). Various PFGE patterns and sequence types (STs) indicated that these isolates were clonally unrelated. Seven different genetic environments of fosA3 were identified and two new combinations (ISEcp1-blaCTX-M-65-ΔIS903D-IS26-fosA3-orf1-orf2-Δorf3-IS26 and IS26-ISEcp1-blaCTX-M-3-orf477-blaTEM-1-IS26-fosA3-orf1-orf2-Δorf3-IS26) were discovered for the first time. Conjugation experiments were successful for 47 isolates and 33 transconjugants harbored a single plasmid. Plasmids carrying fosA3 belonged to incompatibility group IncFII (17/33), IncI1 (2/33), IncHI2 (3/33), and IncB/O (1/33). F33:A-:B- plasmids carrying blaCTX-M-55, IncHI2/ST3 plasmids carrying blaCTX-M-65, and F2:A-:B-plasmids carrying blaCTX-M-55 were found in E. coli isolates from different provinces. Our results revealed a considerable prevalence of fosA3 gene among CTX-M-producing E. coli with clonal diversity from chickens in China. The transmission of different kinds of plasmids is responsible for the dissemination of fosA3 in chicken farms in China.

A label-free fluorescent direct detection of live Salmonella typhimurium using cascade triple trigger sequences-regenerated strand displacement amplification and hairpin template-generated-scaffolded silver nanoclusters.

The harm of Salmonella typhimurium (S. typhimurium) to public health mainly by the consumption of contaminated agricultural products or water stresses an urgent need for rapid detection methods to help control the spread of S. typhimurium. In this work, an intelligently designed sensor system took creative advantage of triple trigger sequences-regenerated strand displacement amplification and self-protective hairpin template-generated-scaffolded silver nanoclusters (AgNCs) for the first time. In the presence of live S. typhimurium, single-stranded trigger sequences were released from aptamer-trigger sequences complex, initiating a branch migration to open the hairpin template I containing complementary scaffolds of AgNCs. Then the first strand displacement amplification was induced to produce numerous scaffolds of AgNCs and reporter strands which initiated a branch migration to open the hairpin template II containing complementary scaffolds of AgNCs. Then the second strand displacement amplification was induced to generate numerous scaffolds of AgNCs and trigger sequences which initiated the third branch migration and strand displacement amplification to produce numerous scaffolds of AgNCs and reporter strands in succession. Cyclically, the reproduction of the trigger sequences and cascade successive production of scaffolds were achieved successfully, forming highly fluorescent AgNCs, thus providing significantly enhanced fluorescent signals to achieve ultrasensitive detection of live S. typhimurium down to 50 CFU/mL with a linear range from 102 to 107CFU/mL. It is the first report on a fluorescent biosensor for detecting viable S. typhimurium directly, which can distinguish from heat denatured S. typhimurium. And it develops a new strategy to generate the DNA-scaffolds for forming AgNCs.

Various Sequence Types of Escherichia coli Isolates Coharboring blaNDM-5 and mcr-1 Genes from a Commercial Swine Farm in China.

Sixteen different sequence types (STs) of Escherichia coli isolates from a commercial swine farm in China were confirmed to coharbor the carbapenem resistance gene blaNDM-5 and the colistin resistance gene mcr-1 Whole-genome sequencing revealed that blaNDM-5 and mcr-1 were located on a 46-kb IncX3 plasmid and a 32-kb IncX4 plasmid, respectively. The two plasmids can transfer together with a low fitness cost, which might explain the presence of various STs of E. coli coharboring blaNDM-5 and mcr-1.

A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis.

[Detection of Ethoprophos Using SERS Coupled with Magnetic Fe3O4/Ag Composite Materials].

The magnetic Fe3O4/Ag composite materials were synthesized by reducing AgNO3 with sodium citrate in the presence of Fe3O4 which were prepared by co-precipitation firstly. The enrichment and extraction of ethoprophos assembled on Fe3O4/Ag were achieved with the applied magnetic field. The different concentrations of ethoprophos adsorbed on Fe3O4/Ag were analyzed by SERS and it was showed that the trace analysis of ethoprophos had been established, while the enhancement factor of probe molecules on Fe3O4/Ag was 1. 48 X 10(5). The structure and morphology of Fe3O4/Ag were characterized by UV-Vis, EDX and TEM. Compared with Ag, the UV-Vis absorption peak of Fe3O4/Ag shifted from 417 to 369 nm, and the UV-Vis of Fe3O4 almost had no characteristic absorption peak in this region. At the same time, it was showed that the surface properties of Fe3O4/Ag changed with Raman enhancement effect during the aggregation process of Ag around the surface of Fe3O4. Further EDX images of micro area element analysis suggested that the chemical composition of products were Ag, Fe and O while the Cu peak was from the copper mesh. In addition, TEM images indicated that the average particle size of Fe3O4 was between 30 and 60 nm with shape tended to be spherical. And the silver nanoparticles were attached to the Fe3O4 particles and agglomeration occured. Density functional theory calculations which can be applied to qualitative judgment of molecule was carried out to obtain the molecular optimization structure and theoretical Raman spectra. It was found that the stabilized SERS signals were detected under the saturated adsorption equilibrium after 15 min. Finally, Raman response of ethoprophos was achieved with lower than 2 X 10(-8) mol . L-1 , indicatint that the established method had reached the requirements of ethoprophos residues detection and could be used for analysis of sulfur-containing organophosphorus pesticide.

[Construction and rescue of rabies virus mutant strain SRV9].

To construct a rabies virus mutant, the psi region was replaced by the coding region of human cytochrome c gene, and the coding region for cytoplasmic domain of glycoprotein G was deleted in the full-length of genomic cDNA of rabies virus strain SRV9. The mutant plasmid and the plasmids with N, P, L and G structural proteins of wild type SRV9 were co-transfected into BHK-21 cells. It was shown by IFA that there were many specific fluorescence in the BHK-21 cells, and typical rabies virus virions were observed by electronic microscope. These results demonstrated that the mutant rabies virus was successfully rescued. The genetically modified SRV9 stain has promise to provide invaluable experimental tool to develop attenuated live rabies vaccine.

Rheumatologic manifestations of hepatic diseases.

A possible link is suggested between hepatic diseases and rheumatic disease. Polyarthralgia and polyarthritis may be seen during the prodromal period of acute viral hepatitis, especially in hepatitis B virus (HBV). The symptoms of arthritis, mild, localized or generalized, mostly involve the small joints of hands. Joint symptoms frequently precede the onset of jaundice, no residual joint deformities. Circulating immune complexes are believed to play a causative role in the development of vasculitis and arthritis. Hemochromatosis is an antosomal recessive disorder of iron. About 43%-81% of patients with hemochromatosis have arthritis. The common extrahepatic manifestations of autoimmune hepatitis are arthralgia and skin rash. The reported prevalence of symptomatic inflammatory arthropathy in patients with primary biliary cirrhosis ranges from 4% to 50%. Skeletal involvement with Wilson's disease is common. Such patients may complain of pain and stiffness, mainly in the knee, wrist, or other large joints. Shwachman's syndrome is a disorder of pancreatic exocrine. Symmetric bone lesions have been reported in 10% to 15% of patients. They are involved predominantly at the femoral neck. Rheumatic symptoms are seen in one third of adult patients with cystic fibrosis and arthritis in 2.5% to 12% of patients. The arthritis caused by pancreatic panniculitis is usually symmetrical and involves the small joints of the hand, wrist, and feet, but may involve such larger joints as the elbow, ankle, and knee.