Clonal lineage and biofilm growth shape cefiderocol activity in Acinetobacter baumannii from oncology patients Ilaria Cavallo, Francesca Sivori, Mauro Truglio, Massimo Francalancia, Elva Abril, et al. Frontiers in Cellular and Infection Microbiology, 2026 Introduction Acinetobacter baumannii is a leading cause of healthcare-associated infections in immunocompromised patients and frequently exhibits multidrug resistance. Cefiderocol, a siderophore cephalosporin, is among the few remaining therapeutic options for infections caused by carbapenem-resistant A. baumannii (CRAB); however, its activity may differ by clonal lineage and can be further compromised in the biofilm state. This study investigates genomic features and cefiderocol efficacy against planktonic and biofilm-associated forms of oncology-derived A. baumannii isolates. Methods Twenty-five non-duplicate, consecutive clinical isolates of A. baumannii from oncology patients underwent whole-genome sequencing and multilocus sequence typing. Cefiderocol activity was quantified in planktonic and biofilm-associated states using minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) assays. Results Ten sequence types were identified, with the high-risk sequence type 2 (ST2) clone accounting for 56% (14/25) of isolates. ST2 strains showed significantly higher resistance to aminoglycosides, carbapenems, and fluoroquinolones than non-ST2 (NST) strains. The carbapenemase gene bla OXA−23 was detected exclusively in ST2. Colistin and cefiderocol were the most active agents overall. ST2 strains showed higher cefiderocol MBC values than NST strains. However, avibactam significantly reduced cefiderocol MBC in ST2, consistent with class D β-lactamases activity. ST2 and NST isolates exhibited comparable distributions of iron acquisition genes and similar CAS-detected siderophore activity under the assay conditions tested. Cefiderocol activity was significantly reduced in biofilms relative to planktonic cells (median MBEC 2 µg/ml versus median MBC 0.5 µg/ml). NST exhibited higher MBEC/MBC ratios than ST2 isolates, indicating greater biofilm-associated tolerance to cefiderocol. Discussion Collectively, these data associate the predominance of oncology-derived ST2 with bla OXA-23 carriage and higher cefiderocol bactericidal thresholds and show that cefiderocol activity is consistently reduced in the biofilm state. Future studies integrating functional measures of iron acquisition and β-lactamase activity will be needed to define the determinants of cefiderocol efficacy across lineages and growth states.
Microbial Profiling of Buffalo Mozzarella Whey and Ricotta Exhausted Whey: Insights into Potential Probiotic Subdominant Strains Andrea Bonfanti, Romano Silvestri, Ettore Novellino, Gian Carlo Tenore, Elisabetta Schiano, et al. Microorganisms, 2025 Buffalo mozzarella cheese whey (CW) and ricotta cheese exhausted whey (RCEW) are valuable by-products of the Mozzarella di Bufala Campana PDO production chain. This study characterized their microbial communities using an integrated culture-dependent and -independent approach. Metabarcoding analysis revealed that the dominance of lactic acid bacteria (LAB), including Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus, alongside diverse heat-resistant yeasts such as Cyberlindnera jadinii. Culture-based isolation identified subdominant lactic acid bacteria strains, not detected by sequencing, belonging to Leuconostoc mesenteroides, Enterococcus faecalis, and Enterococcus durans. These strains were further assessed for their probiotic potential. E. faecalis CW1 and E. durans RCEW2 showed tolerance to acidic pH, bile salts, and lysozyme, as well as a strong biofilm-forming capacity and antimicrobial activity against Bacillus cereus and Staphylococcus aureus. Moreover, bile salt resistance suggests potential functionality in cholesterol metabolism. These findings support the potential use of CW and RCEW as reservoirs of novel, autochthonous probiotic strains and underscore the value of regional dairy by-products in food biotechnology and gut health applications.
Virulence profile of carbapenem-resistant Klebsiella pneumoniae strains by an in vivo model of Galleria mellonella María Guembe, Rama Hafian, Marta Díaz-Navarro, Andrés Visedo, Flavio De Maio, et al. Microbiology Spectrum, 2025 Klebsiella pneumoniae is a significant healthcare-associated pathogen, notable for its diverse virulence and antibiotic resistance profiles. This study aimed to characterize the genotypic and phenotypic diversity of K. pneumoniae isolates and evaluate their virulence using the Galleria mellonella model. Biomass production, metabolic activity, capsule formation, and siderophore production were assessed in 27 K . pneumoniae isolates from hospital-associated infections. Lethality curves were generated using the G. mellonella model, with survival monitored hourly from 16 to 48 hours. The most common sequence types (ST) identified were the high-risk clones ST307 ( N = 10), ST512 ( N = 8), ST101 ( N = 7), and ST661 ( N = 2). These STs were associated with distinct K-locus, including KL102, KL107, KL17, and KL39. Most isolates belonged to the O2afg locus ( N = 18), with the K. pneumoniae carbapenemase genotype detected in 96.3% of strains. None of the isolates were classified as hypervirulent. Phenotypically, ST661 exhibited the highest biomass production despite showing similar metabolic activity to other STs. A positive correlation was observed between biomass and siderophore production, while capsule production was inversely correlated with biomass. In the G. mellonella model, ST661 demonstrated the highest virulence, resulting in 100% mortality by 48 hours, compared to survival rates of 21.4% for ST101, 38.0% for ST307, and 31.2% for ST512. These findings underscore the pathogenic potential of ST661 isolates with enhanced biofilm production. The G. mellonella model may serve as an effective in vivo system for evaluating the virulence of emerging K. pneumoniae lineages. IMPORTANCE We demonstrate that the Galleria mellonella model is a useful tool to analyze the virulence of carbapenem-resistant Klebsiella pneumoniae strains. Our findings highlight the pathogenicity of carbapenem-resistant K pneumoniae isolates, particularly the role of the ST661 that, despite being a rare lineage, harbors the blaVIM gene and is associated with high biofilm production and the highest mortality rates.
The AcrAB efflux pump contributes to the virulence of Enteroaggregative E. coli by influencing the aggregative behavior Martina Laudazzi, Emily Schifano, Francesca Sivori, Ludovica Altieri, Daniela Uccelletti, et al. Frontiers in Cellular and Infection Microbiology, 2025 Multidrug efflux pumps play a major role in the emergence of antibiotic resistance. AcrAB is particularly important among them, as it is the main RND pump in Escherichia coli and other Enterobacteriaceae. In addition to contributing to multidrug resistance, AcrAB also plays a significant role in the virulence of several pathogens. Here, we report that AcrAB contributes to both adhesion to host cells and biofilm formation in EAEC, an enteropathogenic group of E. coli known to cause both acute and persistent diarrhea. EAEC is an emerging pathotype of E. coli characterized by its ability to adhere extensively to epithelial cells in an aggregative manner and to form voluminous biofilms, which favor infection persistence. We found that the deletion of acrB prevents biofilm formation and reduces the export of extracellular DNA (eDNA). By using a specific inhibitor of AcrB, we confirmed the requirement of AcrB transporter activity for biofilm biogenesis. The characteristic aggregative pattern of EAEC is also strongly impaired in the absence of AcrB or in the presence of an efflux-defective AcrB D408A transporter, while it is restored in the ΔacrB strain complemented with acrB. Finally, we show that the EAEC 17-2 ΔacrB derivative is significantly less lethal than the wild type in Caenorhabditis elegans. Complementation with the acrB gene, but not with the acrBD408A allele, fully restores the virulence phenotype after infection. Overall, our results confirm the relevance of the AcrAB efflux pump as a virulence determinant and contribute to understanding the mechanisms adopted by EAEC to form thick biofilms and copious adherence to the epithelial cells, both features enhancing persistence during infections.
Meropenem/vaborbactam activity against carbapenem-resistant Klebsiella pneumoniae from catheter-related bloodstream infections Francesca Sivori, Massimo Francalancia, Mauro Truglio, Ilaria Cavallo, Carmelina Pronesti, et al. Frontiers in Cellular and Infection Microbiology, 2025 IntroductionCarbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant threat in oncology settings due to its multidrug resistance and ability to form biofilms on indwelling medical devices.MethodsThis study investigated the in vitro and in vivo activity of meropenem/vaborbactam (MEV) against two CRKP isolates recovered from catheter-related bloodstream infections in patients undergoing orthopedic oncologic surgery.ResultsWhole-genome sequencing identified the isolates as ST101 and ST307, harboring resistance determinants including blaKPC-3 and blaOXA-1, distributed across IncFII and IncFIB plasmid replicons. Both isolates exhibited extensive resistance to β-lactams, aminoglycosides, and fluoroquinolones but remained susceptible to MEV. Phenotypic assays revealed enhanced biofilm formation and metabolic activity compared to the reference strain Kp ATCC 13883 in the absence of hypervirulence-associated genes. MEV demonstrated bactericidal activity against both planktonic and biofilm-associated cells, with minimum bactericidal concentration (MBC90) and minimum biofilm eradication concentration (MBEC90) values of 0.5/8 μg/ml for CRKP ST101, 0.12/8 μg/ml for CRKP ST307, and 0.25/8 μg/ml for the Kp ATCC 13883 strain. In the Galleria mellonella infection model, MEV significantly improved larval survival following the CRKP challenge.DiscussionThese findings demonstrate that MEV exhibits activity against planktonic and biofilm-associated CRKP cells and highlight the need for further investigation in managing catheter-related bloodstream infections caused by multidrug-resistant K. pneumoniae.
Genomic characterization and antibiotic susceptibility of biofilm-forming Borrelia afzelii and Borrelia garinii from patients with erythema migrans Giorgia Fabrizio, Ilaria Cavallo, Francesca Sivori, Mauro Truglio, Daniela Kovacs, et al. Frontiers in Cellular and Infection Microbiology, 2025 BackgroundBorrelia afzelii and Borrelia garinii are the leading causes of Lyme borreliosis (LB) in Europe. Persistent LB forms may involve biofilms, potentially contributing to antibiotic tolerance.MethodsWhole genome sequencing (WGS) was conducted on 7 B. afzelii and 5 B. garinii isolates from erythema migrans skin biopsies. Biofilms were analyzed for extracellular DNA (eDNA) content and biomass. A phenol red metabolic assay assessed the minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) of amoxicillin, azithromycin, ceftriaxone, and doxycycline.ResultsPhylogenetic analysis revealed B. afzelii and B. garinii formed distinct clades, while B. burgdorferi B31 clustered separately. Core genome analysis showed 38.9% of genes were shared between B. afzelii and B. garinii, decreasing to 26.1% with B. burgdorferi. The cloud genome expanded from 34.4% to 53.4% with the addition of B. burgdorferi. No antimicrobial resistance genes were detected. Surface adhesion gene profiles exhibited significant variation across species, suggesting potential functional differences in host adaptation. B. afzelii and B. garinii species exhibited biofilms, with biomass correlating significantly with eDNA production. MIC values were 0.25 μg/mL (amoxicillin, ceftriaxone), 0.125 μg/mL (azithromycin), and 0.5 μg/mL (doxycycline), with no significant interspecies differences. However, MBIC values were considerably higher: 2 μg/mL (amoxicillin, azithromycin), 16 μg/mL (ceftriaxone), and 32 μg/mL (doxycycline).ConclusionsBiofilms in B. afzelii and B. garinii significantly reduce antibiotic efficacy, particularly ceftriaxone and doxycycline. These in vitro findings highlight the need for targeted therapeutic strategies and suggest biofilms may impact treatment outcomes in LB.
Modulating the skin mycobiome-bacteriome and treating seborrheic dermatitis with a probiotic-enriched oily suspension Mauro Truglio, Francesca Sivori, Ilaria Cavallo, Elva Abril, Valerio Licursi, et al. Scientific Reports, 2024 Seborrheic dermatitis (SD) affects 2–5% of the global population, with imbalances in the skin microbiome implicated in its development. This study assessed the impact of an oily suspension containing Lactobacillus crispatus P17631 and Lacticaseibacillus paracasei I1688 (termed EUTOPLAC) on SD symptoms and the skin mycobiome-bacteriome modulation. 25 SD patients were treated with EUTOPLAC for a week. Symptom severity and skin mycobiome-bacteriome changes were measured at the start of the treatment (T0), after seven days (T8), and three weeks post-treatment (T28). Results indicated symptom improvement post-EUTOPLAC, with notable reductions in the Malassezia genus. Concurrently, bacterial shifts were observed, including a decrease in Staphylococcus and an increase in Lactobacillus and Lacticaseibacillus. Network analysis highlighted post-EUTOPLAC instability in fungal and bacterial interactions, with increased negative correlations between Malassezia and Lactobacillus and Lacticaseibacillus genera. The study suggests EUTOPLAC's potential as a targeted SD treatment, reducing symptoms and modulating the mycobiome-bacteriome composition.
Biofilm-mediated antibiotic tolerance in Staphylococcus aureus from spinal cord stimulation device-related infections Francesca Sivori, Ilaria Cavallo, Mauro Truglio, Lorella Pelagalli, Valerio Mariani, et al. Microbiology Spectrum, 2024 Staphylococcus aureus is a predominant cause of infections in individuals with spinal cord stimulation (SCS) devices. Biofilm formation complicates these infections, commonly requiring both surgical and antibiotic treatments. This study explored the biofilm matrix composition and antimicrobial susceptibility of planktonic and biofilm-growing S. aureus isolates from individuals with SCS-related infections. Whole-genome sequencing (WGS) examined genotypes, virulome, resistome, and the pan-genome structure. The study also analyzed biofilm matrix composition, early surface adhesion, hemolytic activity, and antibiotic-susceptibility testing. WGS revealed genetic diversity among isolates. One isolate, though oxacillin susceptible, contained the mec A gene. The median number of virulence factor genes per isolate was 58. All isolates harbored the biofilm-related ica A/D genes. When assessing phenotypic characteristics, all strains demonstrated the ability to form biofilms in vitro . The antimicrobial susceptibility profile indicated that oxacillin, rifampin, and teicoplanin showed the highest efficacy against S. aureus biofilm. Conversely, high biofilm tolerance was observed for vancomycin, trimethoprim/sulfamethoxazole, and levofloxacin. These findings suggest that S. aureus isolates are highly virulent and produce robust biofilms. In cases of suspected biofilm infections caused by S. aureus , vancomycin should not be the primary choice due to its low activity against biofilm. Instead, oxacillin, rifampin, and teicoplanin appear to be more effective options to manage SCS infections. IMPORTANCE SCS devices are increasingly used to manage chronic pain, but infections associated with these devices, particularly those caused by Staphylococcus aureus , present significant clinical challenges. These infections are often complicated by biofilm formation, which protects bacteria from immune responses and antibiotic treatments, making them difficult to eradicate. Understanding the genetic diversity, virulence, and biofilm characteristics of S. aureus isolates from SCS infections is critical to improving treatment strategies. Our study highlights the need to reconsider commonly used antibiotics like vancomycin, which shows reduced activity against biofilm-growing cells. Identifying more effective alternatives, such as oxacillin, rifampin, and teicoplanin, provides valuable insight for clinicians when managing biofilm-related S. aureus infections in patients with SCS implants. This research contributes to the growing evidence that biofilm formation is crucial in treating device-related infections, emphasizing the importance of tailoring antimicrobial strategies to the biofilm phenotype.
