Digital polymerase chain reaction (dPCR) has emerged as a groundbreaking technology in molecular biology and diagnostics, offering exceptional precision and sensitivity in nucleic acid detection and quantification. This review highlights the core principles and transformative potential of dPCR, particularly in infectious disease diagnostics and environmental surveillance. Emphasizing its evolution from traditional PCR, dPCR provides accurate absolute quantification of target nucleic acids through advanced partitioning techniques. The review addresses the significant impact of dPCR in sepsis diagnosis and management, showcasing its superior sensitivity and specificity in early pathogen detection and identification of drug-resistant genes. Despite its advantages, challenges such as optimization of experimental conditions, standardization of data analysis workflows, and high costs are discussed. Furthermore, we compare various commercially available dPCR platforms, detailing their features and applications in clinical and research settings. Additionally, the review explores dPCR’s role in water microbiology, particularly in wastewater surveillance and monitoring of waterborne pathogens, underscoring its importance in public health protection. In conclusion, future prospects of dPCR, including methodological optimization, integration with innovative technologies, and expansion into new sectors like metagenomics, are explored.

Extracellular vesicles (EVs) are lipid bilayers derived from cell membranes, released by both eukaryotic cells and bacteria into the extracellular environment. During production, EVs carry proteins, nucleic acids, and various compounds, which are then released. While Gram-positive bacteria were traditionally thought incapable of producing EVs due to their thick peptidoglycan cell walls, recent studies on membrane vesicles (MVs) in Gram-positive bacteria have revealed their significant role in bacterial physiology and disease progression. This review explores the current understanding of MVs in Gram-positive bacteria, including the characterization of their content and functions, as well as their interactions with host and bacterial cells. It offers a fresh perspective to enhance our comprehension of Gram-positive bacterial EVs.

Two years have passed since WHO declared a pandemic state for SARS-CoV-2 infection. COVID-19 pathogenesis consists of a first viral phase responsible for early symptoms followed by an inflammatory phase, cytokine-mediated, responsible for late-onset manifestations up to ARDS. The dysregulated immune response has an outstanding role in the progression of pulmonary damage in COVID-19. IL-6, through the induction of pro-inflammatory chemokines and cytokines, plays a key role in the development and maintenance of inflammation, acting as a pioneer of the hyperinflammatory condition and cytokine storm in severe COVID-19. Therefore, drugs targeting both IL-6 and IL-6 receptors have been evaluated in order to blunt the abnormal SARS-CoV-2-induced cytokine release. Sarilumab, a high-affinity anti-IL-6 receptor antibody, may represent a promising weapon to treat the fearsome hyperinflammatory phase by improving the outcome of patients with moderate-to-severe COVID-19 pneumonia. Further prospective and well-designed clinical studies with larger sample sizes and long-term follow-up are needed to assess the efficacy and the safety of this therapeutic approach to achieve improved outcomes in COVID-19.

Streptococcus pyogenes (Group A Streptococcus, GAS) is a Gram-positive pathogen responsible for both superficial and invasive infections (iGAS), with increasing global incidence in recent years. This study aims to characterize the molecular and clinical features of iGAS cases in Bologna and Imola (Italy) between 2022 and 2024. Thirty-five invasive isolates were analyzed through whole-genome sequencing (WGS) to investigate the distribution of emm types, antimicrobial resistance (AMR) genes, and virulence factors. Clinical and epidemiological data were retrospectively collected and analyzed. The majority of cases (80%) were recorded in 2023, predominantly among patients aged over 65 (60%). Bloodstream infections were present in 97.1% of cases, and comorbidities such as diabetes and immunosuppression were common. Empirical antibiotic therapy often involved penicillin/β-lactam inhibitors, while oxazolidinones were the most frequently used in targeted regimens. The in-hospital mortality rate was 20%. Genomic analysis identified emm1, emm12, and emm89 as the most prevalent types, associated with specific virulence profiles and resistance determinants. This study highlights the critical role of emm typing and genomic characterization in understanding the pathogenicity of GAS. These findings contribute to the identification of risk factors for severe outcomes and underscore the need for targeted prevention and treatment strategies in vulnerable populations.

The World Health Organization has identified antimicrobial resistance as a public health emergency and developed a global priority pathogens list of antibiotic-resistant bacteria that can be summarized in the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales species), reminding us of their ability to escape the effect of antibacterial drugs. We previously tested new heteroaryl-ethylene compounds in order to define their spectrum of activity and antibacterial capability. Now, we focus our attention on PB4, a compound with promising MIC and MBC values in all conditions tested. In the present study, we evaluate the activity of PB4 on selected samples of ESKAPE isolates from nosocomial infections: 14 S. aureus, 6 E. faecalis, 7 E. faecium, 12 E. coli and 14 A. baumannii. Furthermore, an ATCC control strain was selected for all species tested. The MIC tests were performed according to the standard method. The PB4 MIC values were within very low ranges regardless of bacterial species and resistance profiles: from 0.12 to 2 mg/L for S. aureus, E. faecalis, E. faecium and A. baumannii. For E. coli, the MIC values obtained were slightly higher (4–64 mg/L) but still promising. The PB4 heteroaryl-ethylenic compound was able to counteract the bacterial growth of both high-priority Gram-positive and Gram-negative clinical strains. Our study contributes to the search for new molecules that can fight bacterial infections, in particular those caused by MDR bacteria in hospitals. In the future, it would be interesting to evaluate the activity of PB4 in animal models to test for its toxicity.

The discovery of compounds with antibacterial activity is crucial in the ongoing battle against antibiotic resistance. We developed two QSAR models to design six novel heteroaryl drug candidates and assessed their antibacterial properties against nine ATCC strains, including Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and also Salmonella enterica and Escherichia coli, many of which belong to the ESKAPE group. We combined PB4, a previously tested compound from published studies, with GC-VI-70, a newly discovered compound, with the best cytotoxicity/MIC profile. By testing sub-MIC concentrations of PB4 with five antibiotics (linezolid, gentamycin, ampicillin, erythromycin, rifampin, and imipenem), we evaluated the combination’s efficacy against the ATCC strains. To assess the compounds’ cytotoxicity, we conducted a 24 h and 48 h 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on colorectal adenocarcinoma (CaCo-2) cells. We tested the antibiotics alone and in combination with PB4. Encouragingly, PB4 reduced the MIC values for GC-VI-70 and for the various clinically used antibiotics. However, it is essential to note that all the compounds studied in this research exhibited cytotoxic activity against cells. These findings highlight the potential of using these compounds in combination with antibiotics to enhance their effectiveness at lower concentrations while minimizing cytotoxic effects.