Linezolid, the first oxazolidinone to be used clinically, is effective in the treatment of infections caused by various Gram-positive pathogens, including multidrug resistant enterococci and methicillin-resistant Staphylococus aureus. It has been used successfully for the treatment of patients with endocarditis and bacteraemia, osteomyelitis, joint infections and tuberculosis and it is often used for treatment of complicated infections when other therapies have failed. Linezolid resistance in Gram-positive cocci has been encountered clinically as well as in vitro, but it is still a rare phenomenon. The resistance to this antibiotic has been, until now, entirely associated with distinct nucleotide substitutions in domain V of the 23S rRNA genes. The number of mutated rRNA genes depends on the dose and duration of linezolid exposure and has been shown to influence the level of linezolid resistance. Mutations in associated ribosomal proteins also affect linezolid activity. A new phenicol and clindamycin resistance phenotype has recently been found to be caused by an RNA methyltransferase designated Cfr. This gene confers resistance to lincosamides, oxazolidinones, streptogramin A, phenicols and pleuromutilins, decrease the susceptibility of S. aureus to tylosin, to josamycin and spiramycin and thus differs from erm rRNA methylase genes. Research into new oxazolidinones with improved characteristics is ongoing. Data reported in patent applications demonstrated that some oxazolidinone derivatives, also with improved characteristics with respect to linezolid, are presently under study: at least three of them are in an advanced phase of development.

The aim of our study was to trace the dynamic changes of hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) lineages in Italy, comparing the genotypic backgrounds of contemporary isolates over a period of 17 years, with those of a sample of early MRSA strains from 1980. In total, 301 non-repetitive MRSA clinical isolates, recovered from 19 Italian hospitals between 1990 and 2007 were selected and analyzed for their antibiotic resistance, typed by PFGE and SCCmec, grouped into clonal-types and further characterized using Multi Locus Sequence Typing (MLST). A sample of fifteen early MRSA strains from 1980 was also used for comparison. The most interesting feature was the recent increase of ST228-MRSA-I (formerly the Italian clone; PFGE E) over the period 2000–2007 (57%), when compared to the period 1990–1999 (29%), and its stability to date, associated with a decrease of the highly epidemic ST247-MRSA-IA (formerly the Iberian clone; PFGE A), (23% from 1990 to 1999, 6% from 2000 to 2007). ST1-MRSA-I (1 out of 2 strains carrying ccr A₂B₂), ST8-MRSA-I (4 strains), ST15-MRSA-I (1 out of 4 carrying ccr A₂B₂) and ST30-MRSA-I (2 out of 5 carrying no ccr AB-types and ccr C) were the predominant earliest STs among the MRSA strains in 1980. A temporal shift in the susceptibility levels to glycopeptides was observed: strains with vancomycin MIC of ≥ 2 mg/L increased from 19.4% to 35.5%. In conclusion, we describe the alternation of MRSA clones that occurred in hospitals from 1990 to 2007 and the increase of the glycopeptide MIC levels, reflecting a worldwide trend. We document the detection of ST1, ST8, ST15 and ST30 in the 1980 isolates; we hypothesize their possible latency and their appearance as the current CA-MRSA clones.

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.

Bacterial internalization is a strategy that non-intracellular microorganisms use to escape the host immune system and survive inside the human body. Among bacterial species, Staphylococcus aureus showed the ability to interact with and infect osteoblasts, causing osteomyelitis as well as bone and joint infection, while also becoming increasingly resistant to antibiotic therapy and a reservoir of bacteria that can make the infection difficult to cure. Despite being a serious issue in orthopedic surgery, little is known about the mechanisms that allow bacteria to enter and survive inside the osteoblasts, due to the lack of consistent experimental models. In this review, we describe the current knowledge about S. aureus internalization mechanisms and various aspects of the interaction between bacteria and osteoblasts (e.g., best experimental conditions, bacteria-induced damages and immune system response), focusing on studies performed using the MG-63 osteoblastic cell line, the best traditional (2D) model for the study of this phenomenon to date. At the same time, as it has been widely demonstrated that 2D culture systems are not completely indicative of the dynamic environment in vivo, and more recent 3D models—representative of bone infection—have also been investigated.

The Coronavirus Disease 19 (COVID-19) pandemic has caused an unexpected death toll worldwide. Even though several guidelines for the management of infectious corpses have been proposed, the limited number of post-mortem analyses during the pandemic has led to inaccuracies in the counting of COVID-19 deaths and contributed to a lack of important information about the pathophysiology of the SARS-CoV-2 infection. Due to the impossibility of carrying out autopsies on all corpses, the scientific community has raised the question of whether confirmatory analyses could be performed on exhumed bodies after a long period of burial to assess the presence of SARS-CoV-2 RNA. Post-mortem lung samples were collected from 16 patients who died from COVID-19 infection and were buried for a long period of time. A custom RNA extraction protocol was developed to enhance extraction of viral RNA from degraded samples and highly sensitive molecular methods, including RT-qPCR and droplet digital PCR (ddPCR), were used to detect the presence of SARS-CoV-2 RNA. The custom extraction protocol developed allowed us to extract total RNA effectively from all lung samples collected. SARS-CoV-2 viral RNA was effectively detected in all samples by both RT-qPCR and ddPCR, regardless of the length of burial. ddPCR results confirmed the persistence of the virus in this anatomical niche and revealed high viral loads in some lung samples, suggesting active infection at the time of death. To the best of our knowledge, this is the first study to demonstrate the persistence of SARS-CoV-2 viral RNA in the lung even after a long post-mortem interval (up to 78 days). The extraction protocol herein described, and the highly sensitive molecular analyses performed, could represent the standard procedures for SARS-CoV-2 detection in degraded lung specimens. Finally, the innovative results obtained encourage post-mortem confirmatory analyses even after a long post-mortem interval.