(Background) The diagnosis and the antimicrobial treatment of orthopedic infection are challenging, especially in cases with culture-negative results. New molecular methods, such as next-generation sequencing (NGS), promise to overcome some limitations of the standard culture, such as the detection of difficult-to-grow bacteria. However, data are scarce regarding the impact of molecular techniques in real-life scenarios. (Methods) We included cases of suspected orthopedic infection treated with surgery from May 2021 to September 2023. We combined traditional cultures with NGS. For NGS, we performed a metagenomic analysis of ribosomal 16s, and we queried dedicated taxonomic libraries to identify the species. To avoid false positive results, we set a cut-off of 1000 counts of the percentage of frequency of reads. (Results) We included 49 patients in our study. Our results show the presence of bacteria in 36/49 (73%) and 29/49 (59%) cases studied with NGS and traditional cultures, respectively. The concordance rate was 61%. Among the 19/49 discordant cases, in 11/19 cases, cultures were negative and NGS positive; in 4/19, cultures were positive and NGS negative; and in the remaining 4/19, different species were detected by traditional cultures and NGS. (Conclusions) Difficult-to-grow microorganisms, such as slow-growing anaerobic bacteria, were better detected by NGS compared to traditional culture in our study. However, more data to distinguish between true pathogens and contaminants are needed. NGS can be an additional tool to be used for the diagnosis of orthopedic infections and the choice of appropriate antimicrobial therapy.

Background/Objectives: Carbapenem-resistant Enterobacteriaceae (CRE) are types of bacteria that need urgent attention globally. Active surveillance programs at hospitals are essential for the early identification of CRE carriers and the timely adoption of infection control measures. We aimed to analyze the epidemiology of CRE identified by multiplex RT-PCR in rectal swabs of patients upon admission to high-risk wards and to compare data obtained from both molecular and culture CRE screening. Methods: A total of 2861 rectal swabs, prospectively collected within 12–24 h of admission, underwent molecular screening for identification of K. pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron-mediated metallo-β-lactamase (VIM), imipenemase (IMP), and OXA-48 (Allplex^TM Entero-DR Assay). Only samples that tested positive or invalid underwent culture testing (Agar MacConkey and CHROMID^® CARBA plates, bioMérieux, Craponne, France). Results: A total of 118 out of 2861 (about 4%) were positive for at least one carbapenem-resistant gene by a molecular approach (MA), with KPC, NDM, and VIM having the highest prevalence. Culture testing confirmed the presence of carbapenemase in 89 samples (75.4%), showing a disagreement rate of about 25% between the two methods, which, unfortunately, rises up to 60% for VIM. The dominant bacterial species were K. pneumoniae and E. coli (MALDI-TOF mass spectrometry). Conclusions: Our data underlined the need for the molecular screening of CRE carriers in order to implement active surveillance protocol in critical care settings and to improve infection control measures.

Bacterial infections pose significant global health challenges, often underestimated due to difficulties in accurate diagnosis, especially when culture-based diagnostics fail. This study assesses the effectiveness of 16S-based metagenomic next generation sequencing (NGS) for identifying pathogens in culture-negative clinical samples across various medical settings. Overall, 48% of samples were collected from orthopedics, 15% from neurosurgery, and 12% in cardiac surgery, among others. The detection rate of monomicrobial infections was 68.6%, and 5.7% for polymicrobial infections. In addition, NGS detected bacteria in all samples from the lungs, head and neck, and eye specimens. Cutibacterium acnes (11%, 12/105) was the most frequent microorganism, followed by Staphylococcus epidermidis (10.4%, 11/105), and Staphylococcus aureus (9.5%, 10/105). In conclusion, 16S-targeted metagenomic sequencing enhances pathogen detection capabilities, particularly in instances where traditional cultures fail. By the combination of NGS and bacterial cultures, microbiologists might provide a more accurate diagnosis, guiding more effective treatments and potentially reducing healthcare costs associated with empirical treatments.

Bacterial meningitis remains a critical public health issue globally due to its high morbidity and mortality. Understanding regional epidemiological trends is essential to inform vaccination strategies and public health interventions. This observational, retrospective study analyzed cerebrospinal fluid (CSF) isolates collected from 731 confirmed cases of bacterial meningitis between 2014 and 2024 in Lombardy, Italy. Pathogen identification and serotyping of Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), and Haemophilus influenzae (HI) were conducted using culture-based and molecular techniques. Trends were assessed across age groups and time using Kruskal–Wallis and chi-square tests. Results: SP was the predominant pathogen (78.4%), followed by NM (13.0%) and HI (8.6%). Significant temporal variation was observed for SP and NM, while HI trends remained stable. The impact of COVID-19-related restrictions was evident in a reduction in cases during 2020–2021. SP serotypes 3 and 8, HI non-typeable strains, and NM serogroup B were most frequent. No major shifts in serotype distribution were observed. Long-term surveillance data from Lombardy underscore the dominance of vaccine-targeted serotypes, ongoing circulation of resilient clones, and post-pandemic epidemiological shifts. These findings support continuous surveillance and inform vaccine strategy adjustments at the regional and national levels.