Molecular Characterization and Potential Host-switching of Swine Farm associated Clostridioides difficile ST11.

OBJECTIVE: To conduct molecular prevalence and genetic polymorphism analysis of 24 Swine Farm associated C. difficile ST11 strains, in addition to other representative sequenced ST strains. METHODS: The collected C. difficile strains underwent whole genome sequencing and bioinformatic analysis using the illumina NovaSeq platform, SPAdes, Prokka, MOB-suite, and FastTree. Virulence and antibiotic resistance genes were identified through NCBI Pathogen Database. Cytotoxicity tests were conducted on HT-29 cells and Vero cells to verify the function of toxin A and toxin B. RESULTS: The most prevalent resistance genes in ST11 were found to be against ß-lactamases, aminoglycosides, and tetracycline. A C. difficile isolate (strain 27) with tcdA deletion and high antibiotic resistance genes was far apart from other swine farm associated ST11 isolates in the phylogenetic branch. The remarkable genetic similarity between animal and human C. difficile strains suggests potential transmission of ST11 strains between animals and humans. The plasmid replicon sequences repUS43 were identified in all ST11 strains except one variant (strain 27), and 91.67% (22/24) of these were assessed by MOB-typer as having mobilizable plasmids. CONCLUSION: Swine farm associated C. difficile ST11 carried fewer virulence genes than ST11 strains collected from NCBI database. It is critical to monitor the evolution of C. difficile strains to understand their changing characteristics, host-switching, and develop effective control and prevention strategies.

A critical role of outer membrane vesicles in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: This study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). METHODS: The data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro. RESULTS: A total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the "enzyme" group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem. CONCLUSIONS: CRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.

Phenotypic and genetic characterization of hypervirulent Klebsiella pneumoniae in patients with liver abscess and ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) and pyogenic liver abscess (PLA) due to Klebsiella pneumoniae infection can trigger life-threatening malignant consequences, however, there are few studies on the strain-associated clinical pathogenic mechanisms between VAP and PLA. A total of 266 patients consist of 129 VAP and 137 PLA were included for analysis in this study. We conducted a comprehensive survey for the two groups of K. pneumoniae isolates, including phenotypic experiments, clinical epidemiology, genomic analysis, and instrumental analysis, i.e., to obtain the genomic differential profile of K. pneumoniae strains responsible for two distinct infection outcomes. We found that PLA group had a propensity for specific underlying diseases, especially diabetes and cholelithiasis. The resistance level of VAP was significantly higher than that of PLA (78.57% vs. 36%, P < 0.001), while the virulence results were opposite. There were also some differences in key signaling pathways of biochemical processes between the two groups. The combination of iucA, rmpA, hypermucoviscous phenotype, and ST23 presented in K. pneumoniae infection is more important and highly prudent for timely treatment. The present study may contribute a benchmark for the K. pneumoniae clinical screening, epidemiological surveillance, and effective therapeutic strategies.

Immunological evaluation of patients with Alzheimer's disease based on mitogen-stimulated cytokine productions and mitochondrial DNA indicators.

BACKGROUND: Based on its objective characteristics, laboratory markers have always been the research direction of clinical diagnosis and assessment of mental disorders including Alzheimer's disease. METHODS: MTT Colorimetric Assay, ELISA, and quantitative PCR were used to investigate the responsiveness of peripheral blood mononuclear cells (PBMCs) to mitogen Lipopolysaccharides (LPS) and Phytohemagglutinin (PHA), PBMCs genomic methylation and hydroxymethylation levels, nuclear DNA and mitochondrial DNA damage, respiratory chain enzyme activities, and circulating cell-free mitochondrial DNA levels were detected in 90 patients with Alzheimer's disease. RESULTS: In the Alzheimer's disease group, LPS stimulated PBMCs viability, TNF-α secretion, PHA stimulated IL-10 secretion, genomic DNA methylation levels, circulating cell-free mitochondrial DNA copies, citrate synthase activity were reduced compared to the control; while the LPS stimulated PBMCs IL-1α secretion, PHA stimulated IL-1α and IFN-γ secretion, plasma IL-6 and TNF-α, mitochondrial DNA damages were increased compared to the control. CONCLUSIONS: The reactivity of peripheral blood mononuclear cells to mitogens, mitochondrial DNA integrity characteristics, and cell-free mitochondrial DNA copies may be used as candidate laboratory biomarkers to help clinical management of Alzheimer's disease.

Serum BAFF and APRIL levels in patients with autoimmune hemolytic anemia and their clinical significance.

B cell-activating factor of the tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) play crucial roles in B cell development, survival, and antibody production. Autoimmune hemolytic anemia (AIHA) is an acquired autoimmune disease that occurs when antibodies target autologous red blood cells. Here, we analyzed the serum levels of BAFF and APRIL and their respective clinical associations in patients with AIHA. Serum BAFF and APRIL levels in patients with AIHA were significantly higher (P < 0.01) than in healthy individuals. Serum BAFF and APRIL levels were significantly augmented in patients with lower hemoglobin levels (hemoglobin was <8 g/dL) and higher LDH activity (LDH > 480 IU/mL). Glucocorticoid treatment dramatically reduced serum levels of BAFF and APRIL. Thus, serum BAFF and APRIL levels may reflect the clinical activity of this disease. Our results indicate that analysis of serum concentrations of BAFF and APRIL potentially represents a useful tool for the assessment of AIHA disease activity and progression.

BAFF level increased in patients with autoimmune hemolytic anemia.

INTRODUCTION: BAFF (B-cell activating factor of the TNF family), an important regulator of B-cell, has been observed to be over-expressed in a variety of autoimmune diseases. Autoimmune hemolytic anemia (AIHA) is an acquired autoimmune disease occurred when antibodies directed against autologous red blood cells. We assessed serum levels of BAFF in AIHA patients with different serological characteristics. METHODS: Serum BAFF levels were measured in 44 AIHA patients with different direct antiglobulin test (DAT) results and 25 healthy controls. The correlation of BAFF expression with DAT results and serological characteristics was assessed. RESULTS: Serum levels of BAFF in AIHA patients were significantly higher than in healthy subjects (AIHA: 1382.7 ± 1412.8 pg/ml, healthy control: 725.0 ± 415.7 pg/ml, P = 0.0057). Serum BAFF levels were significantly higher in patients with IgG(+)C3(+) or IgG(+) than healthy controls (DAT: negative) (P = 0.012, 0.004, respectively). No significant correlations were presented between serum BAFF levels and four serological parameters: hemoglobine, percentage of reticulocyte, total serum bilirubin, and lactate dehydrogenase. CONCLUSIONS: AIHA patients present higher serum BAFF levels than healthy controls, especially for those of IgG(+)C3(+) DAT result. This might lead to a new approach of AIHA treatment.