Stefania Stefani

The mechanisms of action and resistance of cefiderocol (FDC) in Acinetobacter baumannii are still not fully elucidated, but iron transport systems have been evoked in its entry into the cell to reach the penicillin-binding proteins (PBPs). To capture the dynamics of gene expression related to FDC action in various conditions, we report on the genomic and transcriptomic features of seven A. baumannii strains with different FDC susceptibility, focusing on the variants in genes associated with β-lactam resistance and the expression of the siderophore biosynthesis and transport systems acinetobactin and baumannoferrin. We also investigated the expression of the TonB energy transduction system (ETS) and siderophore receptors piu A and pir A. The four clinical samples belonged to the same clonal complex (CC2), and the two strains with the highest FDC MICs showed peculiar variants in PBP2 and amp C. Similarly, the two clinical strains with the lowest MICs shared variants in an outer membrane protein as well as amp C. Gene expression analyses highlighted the up-regulation of the acinetobactin and baumannoferrin genes in response to iron depletion and a down-regulation in the presence of high iron concentrations. In response to FDC, gene expression seemed strain-dependent, probably due to the different metabolic features of each strain. Overall, FDC activates the ETS, confirming the active import of the drug; baumannoferrin, more than acinetobactin, appeared stimulated by FDC in an iron-depleted medium. In conclusion, iron transport systems play a clear role in the FDC uptake, and their expression likely contributes to MIC variation together with β-lactam resistance determinants. IMPORTANCE Acinetobacter baumannii poses a threat to healthcare due to its ability to give difficult-to-treat infections as a consequence of our shortage of antibiotic molecules active on this multidrug-resistant bacterium. Cefiderocol (FDC) represents one of the few drugs active on A. baumannii, and to preserve its activity, this study explored the transcriptomic and genomic features of seven strains with varying susceptibility to FDC. Transcriptomic analyses revealed the different effects of FDC on iron transport systems, promoting mainly baumannoferrin expression—thus more likely related to FDC entry—and the energy transduction systems. These findings suggest that not all iron transport systems are equally involved in FDC entry into A. baumannii cells. Finally, mutations in PBPs and β-lactamases may contribute to the resistance onset. Overall, the study sheds light on the importance of iron availability and metabolic differences in FDC resistance, offering insights into understanding the evolution of resistance in A. baumannii strains.

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.

Background: Vancomycin-intermediate Staphylococcus aureus (VISA) emerges typically in the healthcare-associated methicillin-resistant S. aureus and more rarely in community-acquired S. aureus (CA-MRSA). VISA is a serious concern for public health due to its association with persistent infections, the failure of vancomycin treatment, and poor clinical outcomes. Currently, the burden of VISA is somewhat high, even though vancomycin is the mainstay treatment for severe MRSA infections. The molecular mechanisms of reduced glycopeptide susceptibility in S. aureus are constantly under investigation but have still not yet been fully characterized. Methods: Our goal was to investigate the reduced glycopeptide susceptibility mechanisms emerging in a VISA CA-MRSA versus its vancomycin-susceptible (VSSA) CA-MRSA parents in a hospitalized patient undergoing glycopeptide treatment. Comparative integrated omics, Illumina MiSeq whole-genome sequencing (WGS), RNA-Seq, and bioinformatics were performed. Results: Through a comparison of VISA CA-MRSA vs. its VSSA CA-MRSA parent, mutational and transcriptomic adaptations were found in a pool of genes involved, directly or indirectly, in the biosynthesis of the glycopeptide target conferring or supporting the VISA phenotype, and its cross-resistance with daptomycin. This pool included key genes responsible for the biosynthesis of the peptidoglycan precursors, i.e., D-Ala, the D-Ala-D-Ala dipeptide termini of the pentapeptide, and its incorporation in the nascent pentapeptide, as key targets of the glycopeptide resistance. Furthermore, accessory glycopeptide-target genes involved in the pathways corroborated the key adaptations, and thus, supported the acquisition of the VISA phenotype i.e., transporters, nucleotide metabolism genes, and transcriptional regulators. Finally, transcriptional changes were also found in computationally predicted cis-acting small antisense RNA triggering genes related both to the key or accessory adaptive pathways. Conclusion: Our investigation describes an adaptive resistance pathway acquired under antimicrobial therapy conferring reduced glycopeptide susceptibility in a VISA CA-MRSA due to a comprehensive network of mutational and transcriptional adaptations in genes involved in pathways responsible for the biosynthesis of glycopeptide’s target or supporters of the key resistance path.