Search Results (84)

Background: Salmonella is a bacterium that causes foodborne infections. This study characterized two strains isolated from cheese and beef in Brazil using whole-genome sequencing (WGS). Objectives: We evaluated their antimicrobial resistance profiles, virulence factors, plasmid content, serotypes and phylogenetic relationships. Methods: DNA was extracted and sequenced on the NovaSeq 6000 platform; the pangenome was assembled using the Roary tool; and the phylogenetic tree was constructed via IQ-TREE. Results and Discussion: For contextualization and comparison, 3493 Salmonella genomes of Brazilian origin from NCBI were analyzed. In our isolates, both strains carried the aac(6′)-Iaa_1 gene, while only Schwarzengrund harbored the qnrB19_1 gene and the Col440I_1 plasmid. Cerro presented the islands SPI-1, SPI-2, SPI-3, SPI-4, SPI-5 and SPI-9, while Schwarzengrund also possessed SPI-13 and SPI-14. Upon comparison with other Brazilian genomes, we observed that Cerro and Schwarzengrund represented only 0.40% and 2.03% of the national database, respectively. Furthermore, they revealed that Schwarzengrund presented higher levels of antimicrobial resistance, a finding supported by the higher frequency of plasmids in this serovar. Furthermore, national data corroborated our findings that SPI-13 and SPI-14 were absent in Cerro. A virulence analysis revealed distinct profiles: the cdtB and pltABC genes were present in the Schwarzengrund isolates, while the sseK and tldE1 family genes were exclusive to Cerro. The results indicated that the sequenced strains have pathogenic potential but exhibit low levels of antimicrobial resistance compared to national data. The greater diversity of SPIs in Schwarzengrund explains their prevalence and higher virulence potential. Conclusions: Finally, the serovars exhibit distinct virulence profiles, which results in different clinical outcomes. Full article

The evolving understanding of asthma and COPD pathomechanisms led to this study examining chronic obstructive lung diseases’ impact on cognitive decline—a growing concern in aging populations. We explored whether subunits of key inflammatory regulators NF-κB, c-Rel (neuroprotective), and p65 (neurodegenerative), are linked to cognitive impairment. A pilot study with an explorative design across three groups (asthma, COPD, and control) included 78 patients. Participants underwent assessments via 16 questionnaires (covering demographics, quality of life, disease control, and cognitive and psychiatric evaluations), spirometry, and blood sampling to measure c-Rel and p65 mRNA expression. While both c-Rel and p65 are NF-κB subunits, their expression levels differ independently. Median c-Rel expression was highest, and p65 lowest, in the group with the best cognitive function (control). The most notable correlations for both markers with PKA, CREB, MMSE, and HAM-D were in COPD. The significant association between p65 and the Clock-Drawing Test, without a corresponding link to MMSE, may indicate that a future correlation between p65 and cognitive decline, as assessed by CDT, is likely to emerge. Full article

Background/Objective. Toxin-producing strains of Clostridioides difficile (C. diff) are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalization, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhea with ≥3 unformed stools in 24 h are the target population for C. diff infection (CDI) testing. To present data on the risks of laboratory misdiagnosis in managing CDI. Materials. In two general hospitals, we examined 116 clinical stool specimens from hospitalized patients with acute diarrhea suspected of nosocomial or antibiotic-associated diarrhea (AAD) due to C. diff. Enzyme immunoassay (EIA) tests for the detection of C. diff toxins A (cdtA) and B (cdtB) in stool, automated CLIA assay for the detection of C. diff GDH antigen and qualitative determination of cdtA and B in human feces and anaerobic stool culture were applied for CDI laboratory diagnosis. MALDI-TOF (Bruker) was used to identify the presumptive anaerobic bacterial colonies. The following methods were used as confirmatory diagnostics: the LAMP method for the detection of Salmonella spp. and simultaneous detection of C. jejuni and C. coli, an E. coli Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, and EIEC), API 20E and aerobic stool culture methods. Results. A total of 40 toxigenic strains of C. diff were isolated from all 116 tested diarrheal stool samples, of which 38/40 produced toxin B and 2/40 strains were positive for both cdtA and cdtB. Of the stool samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 were negative for C. diff culture but positive for the following diarrheal agents: Salmonella enterica subsp. arizonae (1/33, LAMP, culture, API 20E); C. jejuni (2/33, LAMP, culture, MALDI TOF); ETEC O142 (1/33), STEC O145 and O138 (2/33, E. coli RT-PCR detection kit, culture); C. perfringens (2/33, anaerobic culture, MALDI TOF); hypermycotic enterotoxigenic K. pneumonia (2/33) and enterotoxigenic P. mirabilis (2/33, culture; PCR encoding LT-toxin). Two of the sixty-six cdtB-positive samples (2/66) showed a similar misdiagnosis when analyzed using the CLIA method. However, the PCR analysis showed that they were cdtB-negative. In contrast, the LAMP method identified a positive result for C. jejuni in one sample, and another was STEC positive (stx1+/stx2+) by RT-PCR. We found an additional discrepancy in the CDI test results: EPEC O86 (RT-PCR eae+) was isolated from a fecal sample positive for GHA enzyme (CLIA) and negative for cdtA and cdtB (CLIA and PCR). However, the culture of C. diff was negative. These findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than C. diff, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EIA card tests for C. diff toxins A and B, which are the most widely used laboratory tests for CDI. Conclusions. CDI presents a significant challenge to clinical practice in terms of laboratory diagnostic management. It is recommended that toxin-only EIA tests should not be used as the sole diagnostic tool for CDI but should be limited to detecting toxins A and B. Accurate diagnosis of CDI requires a combination of laboratory diagnostic methods on which proper infection management depends. Full article

Clostridioides difficile (C. difficile) is a major pathogen responsible for antibiotic-associated diarrhea, frequently observed in hospital settings. Due to the widespread use of antibiotics, the incidence and severity of C. difficile infection (CDI) are rising across the world. CDI is primarily driven by two homologous protein exotoxins, toxin A (TcdA) and toxin B (TcdB). Other putative virulence factors include binary toxin CDT, surface layer proteins, phosphorylated polysaccharides, and spore coat proteins. These C. difficile virulence factors are potential targets for vaccine development. Although several C. difficile vaccines have entered clinical trials, there is currently no approved vaccine on the market. This review outlines the intoxication mechanism during CDI, emphasizing the potential antigens that can be used for vaccine development. We aim to provide a comprehensive overview of the current status of research and development of C. difficile vaccines. Full article

Diffuse large B-cell lymphoma (DLBCL), accounting for 31% of non-Hodgkin lymphomas, remains recalcitrant to conventional therapies due to chemoresistance, metastatic progression, and immunosuppressive microenvironments. We report a novel injectable Fe₃O₄@DMSA@Pt@PLGA-PEG-PLGA hydrogel system integrating magnetothermal therapy (MHT), chemodynamic therapy (CDT), and immunomodulation. Under alternating magnetic fields (AMF), the system achieves rapid therapeutic hyperthermia (50 °C within 7 min) while activating pH/temperature-dual responsive peroxidase (POD) -like activity in Fe₃O₄@DMSA@Pt nanoparticles. Catalytic efficiency under tumor-mimetic conditions was significantly higher than Fe₃O₄@DMSA controls, generating elevated reactive oxygen species (ROS). Flow cytometry revealed 75.9% apoptotic cell death in A20 lymphoma cells at 50 °C, significantly surpassing CDT alone (24.5%). Importantly, this dual mechanism induced immunogenic cell death (ICD) characterized by 4.1-fold CRT externalization, 68% HMGB1 nuclear depletion, and 40.74 nM ATP secretion. This triggered robust dendritic cell maturation (92% CD86⁺/CD80⁺ DCs comparable to LPS controls) and T cell activation (16.9% CD25⁺/CD69⁺ ratio, 130-fold baseline). Our findings validate the therapeutic potential of magnetothermal-chemodynamic synergy for DLBCL treatment, paving the way for innovative multi-mechanism therapeutic strategies against DLBCL with potential clinical translation prospects. Full article

Background: Helicobacter hepaticus (H. hepaticus) has been demonstrated to have clinical relevance to the development of colitis in rodents. H. hepaticus produces cytolethal distending toxins (CDTs), which are identified as the most important virulence factors to the pathogenicity of CDT-producing bacteria in animals. However, the precise relationship between CDTs of H. hepaticus and intestinal barrier dysfunction remains unclear. The objective of the present study was to ascertain the impact of CdtB, the active subunit of CDTs, on the colonic mucosal barrier during H. hepaticus infection. Materials and Methods: We investigated the infection of male BALB/c mice, intestinal organoids, and IEC-6 cell monolayers by H. hepaticus or CdtB-deficient H. hepaticus (ΔCdtB). A comprehensive histopathological examination was conducted, encompassing the assessment of H. hepaticus colonization, the levels of mRNA expression for inflammatory cytokines, the expression levels of tight junction proteins, and the related signaling pathways. Results: The results demonstrate that the presence of ΔCdtB led to a mitigation of the symptoms associated with H. hepaticus-induced colitis, as evidenced by colon length shortening and the colon histological inflammation score. In addition, the levels of pro-inflammatory cytokines were reduced in the ΔCdtB group. Moreover, a downward trend was observed in the phosphorylation levels of STAT3 and nuclear factor-κB (p65). In vitro, the presence of H. hepaticus resulted in a reduction in the expression of tight junction (TJ) markers (ZO-1 and occludin) and an impairment of the F-actin structure in either the intestinal epithelium or intestinal organoids. However, these effects were reversed by CdtB deletion. Concurrently, both ROS levels and apoptosis levels were found to be significantly reduced in cells treated with the ΔCdtB strain. Mechanistically, myosin light chain kinase (MLCK) activation was observed in the H. hepaticus-infected group in vivo, whereas the MLCK inhibitor ML-7 was found to reverse the CdtB-induced alterations in TJ proteins in IEC6 cells. Conclusions: The collective findings demonstrate that CdtB plays a pivotal role in the H. hepaticus-induced colonic mucosal barrier. This is achieved through the regulation of TJs via the MLCK/pMLC2 signaling pathway, which is linked to elevations in oxidative stress and inflammation within intestinal epithelial cells. Full article

Background: In search of indicators for dementia, this study investigated the association of cerebrospinal fluid (CSF) biomarkers and neuropsychological test results with disease stage in patients with early manifestations of dementia. Methods: In 190 consecutive patients with symptoms of dementia, the CSF parameters amyloid-β 1-42 (Aβ1-42), phosphorylated tau protein (pTau), total tau protein (tTau), neuron-specific enolase (NSE), protein S100B (S100B), and Aβ (1-42)/(1-40) ratio (Aβ ratio), as well as the results of the CERAD-Plus test battery supplemented by the Clock Drawing Test (CDT), were analysed. Patients were divided into two groups based on the median duration of reported symptom onset. Results: Most prominent in the early phase of the disease were the relationships between Aβ1-42 and neuropsychological memory subtests, which were absent in the later phase. Less pronounced relationships to memory function were detectable for Aβ ratio and pTau. Conclusions: The results substantiate the relevance of Aβ1-42 for memory deficits and support the amyloid cascade hypothesis for Alzheimer’s dementia (AD). Our data suggest other pathomechanisms for visuospatial impairments in AD. Full article

Clostridioides difficile is a common etiological factor of hospital infections, which, in extreme cases, can lead to the death of patients. Most strains belonging to this bacterium species synthesize very dangerous toxins: toxin A (TcdA) and B (TcdB) and binary toxin (CDT). The aim of this study was to assess the suitability of agarose gel electrophoresis separation of multiplex PCR amplicons to investigate the toxinogenic potential of C. difficile strains. Additionally, the frequency of C. difficile toxin genes and the genotypes of toxin-producing strains were determined. Ninety-nine C. difficile strains were used in the detection of the presence of genes encoding all of these toxins using the multiplex PCR method. In 85 (85.9%) strains, the presence of tcdA genes encoding enterotoxin A was detected. In turn, in 66 (66.7%) isolates, the gene encoding toxin B (tcdB) was present. The lowest number of strains tested was positive for genes encoding a binary toxin. Only 31 (31.3%) strains possessed the cdtB gene and 22 (22.2%) contained both genes for the binary toxin subunits (the cdtB and cdtA genes). A relatively large number of the strains tested had genes encoding toxins, whose presence may result in a severe course of disease. Therefore, the accurate diagnosis of patients, including the detection of all known C. difficile toxin genes, is very important. The multiplex PCR method allows for the quick and accurate determination of whether the tested strains of this bacterium contain toxin genes. Agarose gel electrophoresis is a useful tool for visualizing amplification products, allowing one to confirm the presence of specific C. difficile toxin genes as well as investigate their dissemination for epidemiological purposes. Full article

Escherichia albertii and Escherichia fergusonii are recognized as emerging pathogens with zoonotic potential. Despite their increasing importance, there is a paucity of data on the cytotoxicity of these two pathogens. Therefore, in the present study, we investigated the cytotoxic potentials of the cell-free supernatants from 10 E. albertii and 15 E. fergusonii isolates for their cytotoxic effects on four different cell lines (CHO, Vero, HeLa, and MDCK). All E. albertii isolates (100%) and all but one E. fergusonii (93.33%) were cytotoxic. E. albertii isolates produced similar cytotoxicity titres across the cell lines, whereas the Vero cell was found to be the most sensitive to toxins produced by E. fergusonii (p < 0.05), followed by HeLa and CHO cells. MDCK was the least sensitive cell line to E. fergusonii toxins (p < 0.05). PCR detection of cytotoxicity-associated genes (cdtB, stx1, and stx2) indicated uniform possession of cdtB gene by all E. albertii isolates, while stx1 and stx2 genes were harboured neither by E. albertii, nor E. fergusonii. Taken together, our results provided experimental evidence of the cytotoxic effects of these two emerging pathogens, and Vero cells were identified as an optimal candidate to study the cytotoxic effects of E. albertii and E. fergusonii. Full article

Clostridioides difficile became one of the main causes of nosocomial infections in all clinical settings worldwide, especially among patients undergoing antibiotic therapy. The incidence and severity of C. difficile infections, from mild diarrhea to life-threatening pseudomembranous colitis, correlate with the spread of the hypervirulent binary toxin (CDT)-producing strains. The use of the real-time HRM-PCR method enables the identification of hypervirulent C. difficile strains directly in the diarrheal stool samples of patients suspected of being infected with this bacterium. For this purpose, the cdtA and cdtB genes encoding CDT subunits, as well as the species-specific gluD gene, were detected to identify the presence of this bacterium in the tested samples. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of the established method were also assessed. The obtained results were compared with the results of eazyplex^® C. difficile complete test (AmplexDiagnostics GmbH) based on the LAMP method, used in standard microbiological diagnostics. The values of the assessed diagnostic parameters for the detected genes ranged from 58.82% to 98.85%. The lowest value (58.82%) was obtained for the PPV of cdtB and the highest (98.85%) for the NPV of this gene. The real-time HRM-PCR method enables fast and simple detection of the investigated genes of hypervirulent C. difficile strains and, after careful optimization, may demonstrate high potential for usefulness in routine microbiological diagnostics. Full article

Glaesserella parasuis cytolethal distending toxin (GpCDT) can induce cell cycle arrest and apoptosis. Our laboratory’s previous work demonstrated that GTPase 4b (Rab4b) is a key host protein implicated in GpCDT-induced cytotoxicity. This study investigated the probable involvement of Rab4b in the process. Our study used CRISPR/Cas9 technology to create a Rab4b-knockout cell line. The results showed greater resistance to GpCDT-induced cell cytotoxicity. In contrast, forced Rab4b overexpression increased GpCDT-induced cytotoxicity. Further immunoprecipitation study reveals that GpCDT may bind with Rab4b. In PK-15 cells, GpCDT is transported to the early endosomes and late endosomes, while after knocking out Rab4b, GpCDT cannot be transported to the early endosome via vesicles. Rab4b appears essential for GpCDT-induced cytotoxicity in PK-15 cells. Full article

Objective Campylobacter species are the main causes of foodborne illness worldwide, posing significant threats to public health. This study aimed to investigate the antibiotic resistance and genomic characterization of C. jejuni/C.coli from retail chickens in Beijing. Methods Antimicrobial susceptibility testing was conducted on 126 C. jejuni/C. coli isolated from retail chickens in Beijing, following CLSI protocols. Whole genomes of all isolates were sequenced using the Illumina platform. Results More C. coli (83.82%) showed multi-drug resistance than C. jejuni (8.62%). Genomic analysis demonstrated 42 sequence types (STs) and 12 clonal complexes (CCs), from which CC828 and CC52 were dominant. cdtA, cdtB and cdtC encoding cytotoxic protein were present spontaneously in most C. jejuni but not found in any C. coli isolates. The abundances of antibiotic resistance genes (ARGs) and virulence genes (VGs) in C. jejuni and C. coli were significantly different, with ARGs numbered in C. coli and VGs in C. jejuni. Conclusions High prevalence of multi-drug resistance C. coli and C. jejuni isolated from Beijing chickens were challenging clinical antibiotic usages in the treatment of Campylobacter infection. The surveillance of particular C. jejuni and C. coli STs correlated with higher resistance and virulence needs to be strengthened in the future. Full article

Our research explores the interplay between Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) and the host’s inflammatory response in molar/incisor pattern periodontitis (MIPP). Cdt disrupts phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling, influencing cytokine expression through canonical and non-canonical inflammasome activation as well as nuclear factor-κB (NF-κB) activation, leading to inflammation in MIPP. THP-1 differentiated macrophages (TDMs) exposed to Cdt exhibited an upregulation of pro-inflammatory genes and subsequent cytokine release. We analyzed the ability of a small molecule therapeutic, LGM2605, known for its anti-inflammatory properties, to reduce pro-inflammatory gene expression and cytokine release in Cdt-exposed and Aa-inoculated TDMs. LGM2605’s mechanism of action involves inhibiting NF-κB while activating the Nrf2–transcription factor and antioxidants. Herein, we show that this small molecule therapeutic mitigates Cdt-induced pro-inflammatory cytokine expression and secretion. Our study also further defines Cdt’s impact on osteoclast differentiation and maturation in MIPP. Cdt promotes increased TRAP+ cells, indicating heightened osteoclast differentiation, specific to Cdt’s phosphatase activity. Cathepsin K levels rise during this process, reflecting changes in TRAP distribution between control and Cdt-treated cells. Exploring LGM2605’s effect on Cdt-induced osteoclast differentiation and maturation, we found TRAP+ cells significantly reduced with LGM2605 treatment compared to Cdt alone. Upon LGM2605 treatment, immunocytochemistry revealed a decreased TRAP intensity and number of multinucleated cells. Moreover, immunoblotting showed reduced TRAP and cathepsin K levels, suggesting LGM2605’s potential to curb osteoclast differentiation and maturation by modulating inflammatory cytokines, possibly involving Nrf2 activation. In summary, our research reveals the intricate connections between Cdt, pro-inflammatory cytokines, and osteoclast differentiation, offering novel therapeutic possibilities for managing these conditions. Full article

Background: The postoperative complication rate is 30–64% among patients undergoing muscle-invasive and recurrent high-risk non-muscle-invasive bladder cancer surgery. Preoperative risky alcohol use increases the risk. The aim was to evaluate the accuracy of markers for identifying preoperative risky alcohol. Methods: Diagnostic test sub-study of a randomized controlled trial (STOP-OP trial), based on a cohort of 94 patients scheduled for major bladder cancer surgery. Identification of risky alcohol use using Timeline Follow Back interviews (TLFB) were compared to the AUDIT–C questionnaire and three biomarkers: carbohydrate-deficient transferrin in plasma (P–CDT), phosphatidyl-ethanol in blood (B–PEth), and ethyl glucuronide in urine (U–EtG). Results: The correlation between TLFB and AUDIT–C was strong (ρ = 0.75), while it was moderate between TLFB and the biomarkers (ρ = 0.55–0.65). Overall, sensitivity ranged from 56 to 82% and specificity from 38 to 100%. B–PEth showed the lowest sensitivity at 56%, but the highest specificity of 100%. All tests had high positive predictive values (79–100%), but low negative predictive values (42–55%). Conclusions: Despite high positive predictive values, negative predictive values were weak compared to TLFB. For now, TLFB interviews seem preferable for preoperative identification of risky alcohol use. Full article

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI. Full article