Search Results (65)

Loss of renal function is associated with premature aging of the immune system; however, earlier studies on this topic enrolled patients with reduced GFR and did not distinguish between those with or without proteinuria. Clinical observations suggest that proteinuria alone might also impair immune defense. It is thus an open question whether patients with a urinary albumin-to-creatinine ratio > 300 mg/g (severe albuminuria) independent of their eGFR have an inflammatory risk profile and a disturbed adaptive immune response. This cross-sectional study included 25 patients with severe albuminuria (uACR > 300 mg/g; P-Group), 19 patients with an eGFR < 60 mL/min 1.73 m2 and a uACR < 300 mg/g (G-Group), and 30 patients with adequate kidney function (C-group). All three cohorts have a similar cardiovascular background with arterial hypertension and coronary artery disease. PBMCs from the patients were challenged with the superantigen Staphylococcal enterotoxin B (SEB) as well as with CMV- and SARS-CoV-2-specific antigens. Monocyte subsets and CD86 and HLA-DR expression were determined through flow cytometry. Transcripts of CD28 and IFN-α and -γ were measured by qPCR. Compared to those in the C-group, patients in the G-group showed a higher polyclonal SEB response and had significantly elevated circulating numbers of inflammatory monocytes (subset CD14++CD16+). CD28 transcripts were decreased in the P-group and G-group compared to the C-group, reaching significance for the G-Group. The CMV- and SARS-CoV-2-specific immune response, as measured by the frequency of CD4+69+137+ T and CD8+69+137+ T cells, was comparable in all three groups. Severe albuminuria per se does not alter the antigen-specific immune response; however, patients with reduced GFR, but not those with proteinuria, show inflammatory and polyclonal immune activation. Full article

Staphylococcus aureus is a major bacterial pathogen that can cause clinical infections and foodborne illnesses through the production of 25 exotoxin types. The most frequently implicated toxins in food poisoning outbreaks are Staphylococcal enterotoxins type A–E (SEA-SEE), which are the first enterotoxins discovered. While in vitro detection methods are available to identify the presence of enterotoxins, they cannot distinguish between biologically active and inactive forms of the toxins. Detection of biologically active enterotoxins currently relies on in vivo testing, using the emetic response in kittens or monkeys. Here, we show the development of an in vitro assay to detect the active forms of SEB, a potential biological warfare agent and leading cause of food poisoning, and SEC1, a frequent cause of staphylococcal food poisoning. The novel assay involves the implementation of a genetically engineered Jurkat T-cell line expressing TCR Vβ3, resulting in a dose response of IL-2 production when exposed to active toxin. We also show that at a concentration of 100 ng/mL, the biological activity of SEB is significantly decreased at temperatures over 70 °C, while pasteurization at 63 °C only slightly reduces the biological activity of the toxin. Our studies provide an alternative method to animal testing to determine the presence of active toxins and provide possible inactivation methods of the toxins. Full article

Staphylococcus aureus is a major cause of foodborne intoxication through the production of heat-stable enterotoxins (SEs) and is also an important opportunistic pathogen of humans and livestock. Meat and meat products are major vehicles for this pathogen because their protein-rich composition supports bacterial growth and toxin production. However, the combined effects of temperature and nutrient composition on S. aureus growth and virulence expression under food-relevant conditions remain unclear. In this study, we investigated the interactive effects of temperature and nutritional context on the growth and virulence-associated phenotypes under model food-relevant environments with the reference strain S. aureus FRI-S6. Bacterial growth, biofilm formation, staphylococcal enterotoxins A and B (SEA, SEB), and hemolytic activity were evaluated at 25 °C and 37 °C in brain heart infusion (BHI) medium supplemented with NaCl, glucose, or tryptone to simulate diverse food-relevant conditions. Growth was generally faster at 37 °C, whereas glucose-supplemented cultures at 25 °C reached higher cell densities during prolonged incubation. Biofilm formation increased at 37 °C in BHI and glucose conditions. SEA production was enhanced at 37 °C under NaCl and tryptone, but at 25 °C in glucose-rich conditions. In contrast, SEB production and hemolytic activity were consistently higher at 37 °C, particularly in the presence of tryptone and glucose. These findings demonstrate the strong interaction between temperature and nutrient composition in shaping S. aureus virulence in food environments and provide important insights for food safety risk assessment and highlight practical implications for controlling enterotoxin production in meat products and other foods during storage and processing. Full article

Background/Objectives: Staphylococcus aureus (S. aureus) intramammary infection remains a major global dairy problem due to its contagious nature, its ability to persist and colonize teat/skin and mucosal niches, and the often-limited bacteriological cure achieved with antimicrobial therapy. Beyond udder health, it is relevant to public health because it can enter raw milk chains and serve as a reservoir for antimicrobial resistance determinants that may circulate between dairy animals and humans. Methods: We assessed S. aureus’ ecology in raw ewe milk from 75 sheep farms in Epirus (Greece) by sampling clinically healthy controls (group A) and clinical mastitis cases pre-treatment (group B), followed by resampling at the first post-withdrawal milking after penicillin/streptomycin treatment (group C1—therapeutic protocol 1), oxytetracycline treatment (group C2—therapeutic protocol 2), or enrofloxacin treatment (group C3—therapeutic protocol 3). Results: S. aureus detection was high and comparable across groups (A 23.0%, B 22.0–30.0%, C 20.0–22.0%), and paired analyses showed no significant pre–post shifts in detection/burden within therapeutic protocols (all p > 0.05). Nevertheless, persistence remained evident. The chromosomal gene mecA was detected in S. aureus strains in all groups, ranging from 13.6% in controls to 54.5% post-withdrawal in group C1, and was also present in the pre-treatment group. In paired sampling animals, mecA was mostly stable, with rare emergence or loss. Across antibiotic classes, within-animal resistance transitions were generally uncommon and non-significant (p > 0.05); β-lactam resistance was fully stable (p = 1.00). Descriptively, resistance to protein synthesis inhibitors tended to decline after therapy in protocol 1 and protocol 3, while protocol 3 showed post-treatment gains in fluoroquinolone resistance. By contrast, virulence-associated phenotype traits shifted after therapy: enterotoxigenicity increased post-withdrawal (especially in the C3 group), Staphylococcal Enterotoxin A (SEA) and Staphylococcal Enterotoxin B (SEB) appeared only post-therapy, Staphylococcal Enterotoxin D (SED) increased significantly in paired isolates (p = 0.002), and strong biofilm adherence increased (in C3, p = 1.5 × 10⁻⁵). Conclusions: The detection of S. aureus after therapy suggests that one possibility is that antimicrobial exposure may select for, or otherwise reshape, the residual intramammary population, rather than reliably eliminating it—an outcome that remains clinically relevant for udder health. Moreover, the persistence of mecA/methicillin-resistant Staphylococcus aureus (MRSA)-compatible profiles indicates that milk released to the food chain after withdrawal compliance may still harbor S. aureus with enhanced preservation capacity and significant food safety relevance. Full article

Bacterial toxins, such as Staphylococcal Enterotoxin B (SEB) and Shiga Toxins (STX1, STX2), pose severe public health risks and significant biological threats, demanding rapid and precise qPCR detection. This study reports the initial stages of standardization for uniplex qPCR components, serving as a foundational step toward a future multiplex detection system. The annealing temperature was successfully optimized for the stx1, seb, and 16S rRNA targets, showing high consistency in Cq values and fluorescence intensities at 58.4 °C. The optimized primer ratios ensure maximum amplification efficiency while minimizing potential molecular competition. These optimized assays provide a robust foundation for the multiplex qPCR platform. Once fully validated, this protocol will enhance timely and effective responses, as well as improve preparedness and readiness within the Brazilian biodefense system. Full article

Platelet concentrates (PCs) are used to treat patients with platelet deficiencies. PCs are stored at 20–24 °C under agitation for up to 7 days to maintain platelet functionality, but these conditions are amenable for proliferation of contaminants such as Staphylococcus aureus, posing a risk for transfusion-transmitted infections. We investigated the contribution of staphylococcal enterotoxins (SEs) type G (SEG) and type H (SEH) to platelet activation, cytokine release, microRNA (miRNA) modulation, and in vivo virulence. PCs were inoculated with wildtype S. aureus CBS2016-05 or SE-deficient mutants (Δseg, Δseh, ΔΔsegh) and monitored during storage. Flow cytometry revealed progressive elevation of platelet activation markers CD62P and Annexin V in contaminated PCs, with significantly higher expression in wildtype compared to SE-mutant strains. Cytokine profiling demonstrated that SEs modulate pro- and anti-inflammatory mediators, notably CCL2, TGF-β1, IFN-γ, and TNF-α, implicating SEG in their regulation. Next-generation sequencing and RT-qPCR validation identified transient induction of immune-related microRNAs miR-98-5p, miR-146a-5p, miR-221-3p, miR-320a-3p, with SE-dependent expression patterns. In a silkworm infection model, wildtype S. aureus-contaminated PCs exhibited significantly higher lethality than SE-deficient strains, confirming toxin-mediated virulence. Collectively, these findings reveal that SEs exacerbate platelet activation and immune dysregulation during storage, enhancing bacterial pathogenicity. This study identifies platelet-derived cytokine and miRNA signatures as potential biomarkers of bacterial contamination and underscores the need to mitigate SE-driven platelet dysfunction to improve transfusion safety. Full article

Staphylococcal enterotoxins (SEs), particularly enterotoxin C (SEC), are potent superantigens primarily known for causing food poisoning, but recent studies have highlighted their potential role in immune-mediated intestinal diseases. Despite the widespread use of food preservatives, their influence on SEC production—especially from coagulase-negative staphylococci (CNS)—remains poorly understood. In this study, we evaluated the effects of commonly used preservatives, including sodium chloride, potassium nitrate, and sorbic acid, on the expression and production of SEC₃ and SEC_epi in Staphylococcus aureus and S. epidermidis, respectively. Using ELISA and RT-qPCR, we analyzed toxin levels at both the protein and mRNA levels. Proliferation assays on human PBMCs assessed the mitogenic potential of culture supernatants. While sodium chloride and potassium nitrate did not significantly alter SEC levels or bacterial growth, only sorbic acid at 0.07% consistently inhibited both mRNA expression and protein production of SEC₃ and SEC_epi. Furthermore, supernatants from sorbic acid-treated cultures induced significantly lower PBMC proliferation. These results suggest that even sub-emetic concentrations of enterotoxins may have immunomodulatory effects, and sorbic acid could be a promising agent in mitigating such risks. Full article

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples. Full article

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEM_WT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His_6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His_6×-TCS-ΔNspSEM_WT. Unexpectedly, 4 °C desalting reduced the fusion protein’s molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R–G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H₁₇₂/S₁₇₈/D₂₁₂) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His_6×-TCS-ΔNspSEM_S178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity’s origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R–G bond cleavage. This work establishes a foundation for understanding ΔNspSEM_WT’s TLA. Full article

Lidocaine exhibited anti-inflammatory and immunomodulatory properties. This study aimed to investigate the anti-inflammatory effects of the lidocaine-derived analogs, EI137 and EI341, in a Staphylococcal enterotoxin B (SEB)-induced chronic rhinosinusitis (CRS). A CRS model was established using BALB/c mice via intranasal instillation of SEB. EI137 and EI341 were administered intranasally at 0.5 μg/g and 5 μg/g, respectively. Nasal symptoms and interleukin (IL)-4, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α levels in the nasal lavage fluid (NLF) were assessed. The reverse-transcription polymerase chain reaction was used to identify IFN-γ, IL-4, IL-10, and their transcription factors in the sinonasal mucosa. Histological changes were performed to assess inflammatory cell infiltration, epithelial thickness, and mucus-producing cells. SEB induced significant increases in IL-4, IL-10, and TNF-α levels in NLF and sinonasal mucosa, along with marked inflammatory cell infiltration. Intranasal EI137 and EI341 administration significantly reduced Th2 cytokine and its transcription factor, inflammatory cell infiltration, and mucus-producing cell numbers in the sinonasal mucosa. Further, EI137 suppressed Th1 cytokines, whereas EI341 enhanced Th1 responses. Both compounds promoted regulatory T cell responses, as evidenced by increased IL-10 and Foxp3 mRNA expression. EI137 and EI341 demonstrated potent local anti-inflammatory effects in a SEB-induced CRS model by modulating Th2 and Treg responses. EI137 suppressed Th1 inflammation, whereas EI341 enhanced it. These results indicate that EI137 and EI341 are promising topical agents for Th2-dominant inflammatory diseases, with distinct effects on Th1 immune responses. Full article

Staphylococcus spp. skin colonization is involved in the pathogenesis of atopic dermatitis (AD). While coagulase-positive Staphylococcus aureus strains are known to worsen symptoms, the role of coagulase-negative staphylococci (CoNS) remains controversial. Further research is needed to clarify the pathogenicity of CoNS in AD patients. A study involving 329 children with AD (mean age: 4.89 years) assessed the frequency of staphylococcal colonization on affected skin, along with the toxin-producing properties and antibiotic resistance of isolated strains. Mild AD: Predominantly colonized by CoNS (especially S. epidermidis). Moderate/Severe AD: Showed a significant increase in S. aureus colonization. CoNS (including S. epidermidis) could produce enterotoxins (A, B, C) and toxic shock syndrome toxin-1 (TSST-1), though less frequently than S. aureus strains. In severe AD, the number of toxin-producing CoNS strains (especially enterotoxin A producers) was higher than in mild AD, and the number of non-toxin-producing strains was lower. CoNS exhibited higher resistance rates than S. aureus. Methicillin-resistant S. epidermidis (MRSE): 23.4%. Methicillin-resistant S. aureus (MRSA): 1.27%. CoNS may contribute to AD pathogenesis through toxin production (exacerbating inflammation) and antibiotic resistance (limiting treatment options). Severe AD may involve a synergistic effect between S. aureus and toxin-producing CoNS. Full article

Chronic Rhinosinusitis (CR) is a common inflammatory condition with complex pathophysiology involving multiple interleukins. In times of precision medicine, it is mandatory to cluster our patients to offer the best tailored treatment with the lowest cost possible. Therefore, some triage markers can be used towards this goal, without raising much financial burden. The aim of this study was to identify the association of staphylococcal enterotoxin (SE)-specific IgE of types A, B, C, and TSST-1 (toxic shock syndrome toxin-1); and total IgE (tIgE) and specific IgE for Dermatophagoides pteronyssinus (DP), Dermatophagoides farinae (DF), and Blomia tropicalis (BT) in Brazilian patients with CRSwNP. Thirty-six patients with CSRwNP were analyzed for serum IgE levels: tIgE and specific IgE for: DP, DF, BT, and SE types A, B, C, TSST-1 by ImmunoCAP^®. The mean value of tIgE in SE-specific IgE-positive patients was 767 IU/mL and in house-dust-mite (HDM)-positive patients, the mean tIgE was 319 IU/mL (p < 0.005). A total of 86% of patients who had high tIgE levels but were SE-specific IgE-negative had positive specific IgE for at least one of the HDMs tested. The Fisher exact test statistic value for this association was significant (p < 0.05/p = 0.014). We found an association between high levels of tIgE and SE-specific IgE in patients with CRSwNP, possibly related to local and peripheric polyclonal IgE production. The mean value of tIgE—with a suggested cutoff point of tIgE levels of 767 IU/mL—can be used as a triage biomarker for positive SE-specific IgE in CRSwNP patients. Full article

Leukocidins of Staphylococcus (S.) aureus are bicomponent toxins that form polymeric pores in host leukocyte membranes, leading to cell death and/or triggering apoptosis. Some of these toxin genes are located on prophages and are associated with specific hosts. The genes lukP/Q have been described from equine S. aureus isolates. We examined the genomes, including the lukP/Q prophages, of S. aureus strains belonging to clonal complexes CC1, CC350, CC816, and CC8115. In addition to sequencing, phages were characterised by mitomycin C induction and transmission electron microscopy (TEM). All lukP/Q prophages integrated into the lip2=geh gene, and all included also the gene scn-eq encoding an equine staphylococcal complement inhibitor. The lukP/Q prophages clustered, based on gene content and allelic variants, into three groups. One was found in CC1 and CC97 sequences; one was present mainly in CC350 but also in other lineages (CC1, CC97, CC133, CC398); and a third one was exclusively observed in CC816 and CC8115. Prophages of the latter group additionally included a rare enterotoxin A allele (sea_320E). Moreover, a prophage from a CC522 goat isolate was found to harbour lukP. Its lukF component could be regarded as chimaera comprising parts of lukQ and of lukF-P83. A putative kinase gene of 1095 basepairs was found to be associated with equine strains of S. aureus. It was also localised on prophages. However, these prophages were different from the ones that carried lukP/Q, and three different integration sites of kinase-carrying phages were identified. These observations confirmed the presence of prophage-located important virulence-associated genes in equine S. aureus and that certain prophages might determine the host specificity of the staphylococcal strains they reside in. Full article

This study investigated the toxigenic genes and antimicrobial resistance profiles of Staphylococcus aureus strains isolated from 260 raw milk samples collected from dairy farms in Türkiye. The results indicated that 60.7% of staphylococcal enterotoxin genes (sea, seb, sed, seg, sei, sej, sek, seq, sem, seo, and seu) and 21.4% of the tst and eta genes were positive, with most enterotoxin-positive samples carrying more than one gene. The sec, see, seh, sel, sen, sep, and etb genes were not identified in any samples. The prevalence of antibiotic resistance genes (mecA, blaR, blaI, blaZ, vanA, ermT, tetK, aac/aph, ant, dfrA, tcaR, IS256, and IS257) was high at 89.2%, with bla being the most frequently detected gene (75%). The mecA gene was present in 14.2% of samples, while tcaR was detected in 78.5%. Nevertheless, the mecC was not identified. Disinfectant resistance genes (qacA/B, qacC, qacJ, smr) were detected in 21.4% of the samples. The results of the disk diffusion test showed that 64.2% of strains were resistant to penicillin G and ampicillin, with additional resistance found for cefoxitin, teicoplanin, levofloxacin, norfloxacin, and other antibiotics. These findings highlight a significant public health and food safety risk associated with raw milk due to the presence of S. aureus strains with toxigenic genes and high antimicrobial resistance. Full article

Toxic shock syndrome (TSS) is a rare, life-threatening, toxin-mediated infectious process linked, in the vast majority of cases, to toxin-producing strains of Staphylococcus aureus or Streptococcus pyogenes. The pathophysiology, epidemiology, clinical presentation, microbiological features, management and outcome of TSS are described in this review. Bacterial superantigenic exotoxins induces unconventional polyclonal lymphocyte activation, which leads to rapid shock, multiple organ failure syndrome, and death. The main described superantigenic exotoxins are toxic shock syndrome toxin—1 (TSST-1) and enterotoxins for Staphylococcus aureus and Streptococcal pyrogenic exotoxins (SpE) A, B, and C and streptococcal superantigen A (SsA) for Streptococcus pyogenes. Staphylococcal TSS can be menstrual or nonmenstrual. Streptococcal TSS is linked to a severe group A streptococcal infection and, most frequently, to a necrotizing soft tissue infection. Management of TSS is a medical emergency and relies on early detection, immediate resuscitation, source control and eradication of toxin production, bactericidal antibiotic treatment, and protein synthesis inhibiting antibiotic administration. The interest of polyclonal intravenous immunoglobulin G administration as an adjunctive treatment for TSS requires further evaluation. Scientific literature on TSS mainly consists of observational studies, clinical cases, and in vitro data; although more data on TSS are required, additional studies will be difficult to conduct due to the low incidence of the disease. Full article