The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in , including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant strains constitutes a serious global threat. Carbapenems have been successfully utilized as last-resort antibiotics for the treatment of multidrug-resistant infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work was to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor that may have a significant role in eradicating infections caused by isolates carrying CHDLs.

Acinetobacter baumannii has emerged in the last decade as an important nosocomial pathogen. To identify genes involved in the course of a pneumonia infection, gene expression profiles were obtained from A. baumannii ATCC 17978 grown in mouse infected lungs and in culture medium. Gene expression analysis allowed us to determine a gene, the A1S_0242 gene (feoA), over-expressed during the pneumonia infection. In the present work, we evaluate the role of this gene, involved in iron uptake. The inactivation of the A1S_0242 gene resulted in an increase susceptibility to oxidative stress and a decrease in biofilm formation, in adherence to A549 cells and in fitness. In addition, infection of G. mellonella and pneumonia in mice showed that the virulence of the Δ0242 mutant was significantly attenuated. Data presented in this work indicated that the A1S_0242 gene from A. baumannii ATCC 17978 strain plays a role in fitness, adhesion, biofilm formation, growth, and, definitively, in virulence. Taken together, these observations show the implication of the feoA gene plays in the pathogenesis of A. baumannii and highlight its value as a potential therapeutic target.

In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.

Some d-amino acids can inhibit bacterial growth, biofilm formation and adherence to eukaryotic cells in A. baumannii and P. aeruginosa, and showed a protective effect against infection of alveolar cells with P. aeruginosa. Despite the fact that some considerable protection was observed in mice, survival differences between treated and control groups were not statistically significant.

The pyrrole-imidazoles, a group of alkaloids commonly found in marine sponges belonging to the genus Agelas, display a wide range of biological activities. Herein, we report the first chemical study of the secondary metabolites of the sponge A. dilatata from the coastal area of the Yucatan Peninsula (Mexico). In this study, we isolated eight known alkaloids from an organic extract of the sponge. We used NMR and MS analysis and comparison with existing databases to characterize the alkaloids: ageliferin (1), bromoageliferin (2), dibromoageliferin (3), sceptrin (4), nakamuric acid (5), 4-bromo-1H-pyrrole-2-carboxylic acid (6), 4,5-dibromopyrrole-2-carboxylic acid (7) and 3,7-dimethylisoguanine (8). We also evaluated, for the first time, the activity of these alkaloids against the most problematic multidrug-resistant (MDR) pathogens, i.e., the Gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Bromoageliferin (2) displayed significant activity against P. aeruginosa. Comparison of the antibacterial activity of ageliferins 1–3 (of similar structure) against P. aeruginosa revealed some relationship between structure and activity. Furthermore, in in vitro assays, 2 inhibited growth and biofilm production in clinical strains of P. aeruginosa. Moreover, 2 increased the survival time in an in vivo Galleria mellonella model of infection. The findings confirm bromoageliferin (2) as a potential lead for designing new antibacterial drugs.

Background Pseudomonas aeruginosa may develop resistance to novel cephalosporin/β-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. Objectives To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. Methods Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. Results WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. Conclusions We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/β-lactamase inhibitors in P. aeruginosa.

Background Imipenem/relebactam is a novel carbapenem/β-lactamase inhibitor combination, developed to act against carbapenemase-producing Enterobacterales (CPE). Objectives To assess the in vitro activity of imipenem/relebactam against a Spanish nationwide collection of CPE by testing the susceptibility of these isolates to 16 widely used antimicrobials and to determine the underlying β-lactam resistance mechanisms involved and the molecular epidemiology of carbapenemases in Spain. Materials and methods Clinical CPE isolates (n = 401) collected for 2 months from 24 hospitals in Spain were tested. MIC50, MIC90 and susceptibility/resistance rates were interpreted in accordance with the EUCAST guidelines. β-Lactam resistance mechanisms and molecular epidemiology were characterized by WGS. Results For all isolates, high rates of susceptibility to colistin (86.5%; MIC50/90 = 0.12/8 mg/L), imipenem/relebactam (85.8%; MIC50/90 = 0.5/4 mg/L) and ceftazidime/avibactam (83.8%, MIC50/90 = 1/≥256 mg/L) were observed. The subgroups of isolates producing OXA-48-like (n = 305, 75.1%) and KPC-like enzymes (n = 44, 10.8%) were highly susceptible to ceftazidime/avibactam (97.7%, MIC50/90 = 1/2 mg/L) and imipenem/relebactam (100.0%, MIC50/90 = ≤0.25/1 mg/L), respectively. The most widely disseminated high-risk clones of carbapenemase-producing Klebsiella pneumoniae across Spain were found to be ST11, ST147, ST392 and ST15 (mostly associated with OXA-48) and ST258/512 (in all cases producing KPC). Conclusions Imipenem/relebactam, colistin and ceftazidime/avibactam were the most active antimicrobials against all CPEs. Imipenem/relebactam is a valuable addition to the antimicrobial arsenal used in the fight against CPE, particularly against KPC-producing isolates, which in all cases were susceptible to this combination.

Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa. Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-β-lactamase blaVIM-20 and the narrow-spectrum oxacillinase blaOXA-2 were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the blaOXA-2 β-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin–β-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa.