Invasive group A streptococci infections are increasing worldwide, mainly due to the emm1 lineage M1_UK emergence. Although this variant has recently been described in adult Italian patients, its circulation in children has not yet been established. Characterizing by whole genome sequencing 6 invasive group A streptococci strains isolated between 2022 and 2023, we highlighted M1_UK lineage circulation in pediatric patients in Italy.

DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10⁻³ transconjugants/donor cells.

Candida viswanathii has been sporadically reported in Asia and South America but not in Europe. This study reports the first European outbreak of C. viswanathii in a paediatric hospital, outlining diagnostic challenges and containment measures. Fifteen C. viswanathii isolates were recovered from blood cultures of consecutive pediatric patients admitted to intensive care units between April and August 2025. Identification was performed using MALDI-TOF MS, chromogenic media, Fourier-transform infrared (FT-IR) spectroscopy, and whole-genome sequencing (WGS). Antifungal susceptibility testing was performed by broth microdilution. All isolates were initially misidentified as C. tropicalis by MALDI-TOF MS and undetected by the FilmArray BCID2 panel. WGS confirmed C. viswanathii, and FT-IR analysis revealed clonally related strains, indicating an outbreak. Colonies displayed a distinct deep-blue color on chromogenic CHROMagar™ medium. Elevated fluconazole minimum inhibitory concentrations were observed, while isolates remained susceptible to echinocandins and amphotericin B. A multidisciplinary infection-control response halted transmission within four weeks. This investigation documents the first C. viswanathii outbreak in Europe, highlighting diagnostic limitations of current commercial tools and the need for updated databases. Integration of FT-IR spectroscopy and WGS facilitated outbreak detection and containment, underscoring the importance of advanced diagnostics and surveillance for emerging fungal pathogens.

Cystic fibrosis (CF) is a life-threatening genetic disease characterised by chronic lung infections sustained by opportunistic pathogens such as Pseudomonas aeruginosa. During the chronic long-lasting lung infections, P. aeruginosa adapts to the host environment. Hypermutability, mainly due to defects in the DNA repair system, resulting in an increased spontaneous mutation rate, represents a way to boost the rapid adaptation frequently encountered in CF P. aeruginosa isolates. We selected 609 isolates from 51 patients with CF chronically colonised by P. aeruginosa to study, by full-length genome sequencing, the longitudinal evolution of the bacterium. We recovered at least one hypermutable (mutator) isolate in 57% of patients. By combining genomic information and phenotypic analyses, we followed the evolutionary pathways of the P. aeruginosa mutator strains, identifying their contribution to multi-drug resistance and the emergence of new sub-lineages. By implementing patient clinical data, we observed that mutators preferentially follow a specific evolutionary trajectory in patients with a negative clinical outcome and that maintenance antibiotic polytherapy, based on alternating molecules, apparently reduces the occurrence of hypermutability. Finally, we draw attention to the possibility that modulator-induced changes in the pulmonary environment may be associated with the onset of hypermutability.

Background: Bloodstream infections affect up to 20% of pediatric cancer patients receiving intensive care, contributing significantly to morbidity and mortality, with infection-related mortality rates reported to be as high as 16%. Methods: The identification of microorganisms directly from whole blood is difficult due to several factors, such as interference from host genomic material, low bacterial load, the endogenous components of whole blood and exogenous substances, which can interfere with the identification process. Nevertheless, rapid microbial diagnosis remains of paramount importance in these patients. Results and Conclusion: Here, we present the first case of bacterial pathogen identification directly from whole blood using MALDI-TOF mass spectrometry in an onco-hematological pediatric patient affected by sepsis and admitted to Bambino Gesù Children’s Hospital (IRCCS) in Rome, Italy.

Background/Objectives: Paediatric osteoarticular infections (POAIs) present unique diagnostic and therapeutic challenges. Microbiological culture (MC) is typically time-consuming and lacks sensitivity, especially when patients have received antibiotics. The BIOFIRE^® Joint Infection Panel (BJIP) is a syndromic molecular assay for the direct identification of most pathogens causing POAIs. Methods: We evaluated BJIP in 17 synovial fluids, and then, we retrospectively assessed its utility in 93 off-label specimens (i.e., 25 purulent fluids/biopsies and 68 whole blood samples). All specimens were collected from October 2022 to March 2024 from paediatric patients admitted at the Bambino Gesù Children’s Hospital in Rome. Results: A bacterial pathogen was isolated in only one of 17 synovial fluid cultures, while BJIP identified eight additional microorganisms in MC-negative cases. The most frequently detected pathogen was S. aureus (44.5%, 4/9). BJIP performance in synovial fluids showed an overall positive percentage agreement (PPA) and negative percentage agreement (NPA) of 100% and 88.1%, respectively, compared to MC. All positive results (n/N = 9/17) were considered medically significant, with an increase in NPA to 100%. In purulent fluids/biopsies, BJIP and MC were concordant in 72% of cases (n/N = 18/25), with a per-sample PPA and NPA of 90% and 60%, respectively. For whole blood samples, almost all samples were negative by both methods (i.e., reference blood culture and BJIP), and the molecular test did not enable any further microbiological diagnosis. Conclusions: The BIOFIRE^® Joint Infection Panel rapidly and accurately enabled or excluded a diagnosis of a POAI (~1 vs. 24–96 h for MC), optimising antimicrobial therapy.