OXA-carbapenemases and mutations within PBPs in ST2 carbapenem-resistant A. baumannii: Evaluating the efficacy of cefiderocol and ampicillin-sulbactam combination therapy

Objectives: Carbapenem-resistant Acinetobacter baumannii (A. baumannii; CRAB) isolates represent a serious public health concern. Recently, a novel molecule, the cefiderocol (FDC), has emerged as a promising therapeutic option for CRAB infections. In the present study, we analysed the genomes of five A. baumannii ST2 isolates from four hospitalized patients. All patients were treated with FDC and an ampicillin/sulbactam (AMP/SUL) combination. Methods: Whole-genome sequencing of the five CRAB isolates was performed using an Illumina MiSeq instrument. A detailed bioinformatic analysis was carried out to acquire information about genotyping, antimicrobial resistance genes (ARGs), virulence associated genes (VAGs), single nucleotide polymorphisms (SNPs), and the phylogenetic tree of the five CRAB isolates. Results: Among the five CRAB isolates, only three (Ab.2, Ab.3, and Ab.4) exhibited resistance to FDC. The genomes of all isolates were highly similar, and multilocus sequence typing (MLST) analysis indicated they all belong to sequence type 2 (ST2), corresponding to international clone 2. Phylogenetic analysis suggests that isolates Ab.2, Ab.3, and Ab.4 may share a common ancestor or be linked by a possible transmission event. In contrast, isolates Ab.1 and Ab.5 were more divergent from the other three. Nevertheless, all five isolates harboured the same ARGs and VAGs. The OXA-23, OXA-66, and ADC-25 β-lactamases were detected in all strains. The FDC-non-susceptible isolates showed a K235N/H370Y double mutation within PBP3, along with a G370C substitution in PBP1a. Conclusions: The four clinical cases described in this study represent an important example of the efficacy and good practice of FDC plus AMP/SUL combination in the treatment of critical patients suffering from CRAB infections. © 2025 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates

Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER006684), and the PhD grants SFRH/BPD/111653/2015 and SFRH/BPD/69356/2010

Regional differences in the three-dimensional bone microstructure of the radial head:implications for observed fracture patterns

Introduction: A characterization of the internal bone microstructure of the radial head could provide a better understanding of commonly occurring fracture patterns frequently involving the (antero)lateral quadrant, for which a clear explanation is still lacking. The aim of this study is to describe the radial head bone microstructure using micro-computed tomography (micro-CT) and to relate it to gross morphology, function and possible fracture patterns. Materials and methods: Dry cadaveric human radii were scanned by micro-CT (17 μm/pixel, isotropic). The trabecular bone microstructure was quantified on axial image stacks in four quadrants: the anterolateral (AL), posterolateral (PL), posteromedial (PM) and anteromedial (AM) quadrant. Results: The AL and PL quadrants displayed the significantly lowest bone volume fraction and trabecular number (BV/TV range 12.3–25.1%, Tb.N range 0.73–1.16 mm−1) and highest trabecular separation (Tb.Sp range 0.59–0.82 mm), compared to the PM and AM quadrants (BV/TV range 19.9–36.9%, Tb.N range 0.96–1.61 mm−1, Tb.Sp range 0.45–0.74 mm) (p = 0.03). Conclusions: Our microstructural results suggest that the lateral side is the “weaker side”, exhibiting lower bone volume faction, less trabeculae and higher trabecular separation, compared to the medial side. As the forearm is pronated during most falls, the underlying bone microstructure could explain commonly observed fracture patterns of the radial head, particularly more often involving the AL quadrant. If screw fixation in radial head fractures is considered, surgeons should take advantage of the “stronger” bone microstructure of the medial side of the radial head, should the fracture line allow this

A machine learning approach for mapping susceptibility to land subsidence caused by ground water extraction

Land subsidence is a worldwide threat that may cause irreversible damage to the environment and the infrastructures. Thus, identifying and mapping areas prone to land subsidence with accurate methods such as Land Subsidence Susceptibility Index (LSSI) mapping is crucial for mitigating the adverse impacts of this geohazard. Also, Machine Learning (ML) is now becoming a powerful tool to analyze vast and different datasets such as those necessary for LSSI mapping. In this study, we use the conventional Frequency Ratio (FR) method and ML models to generate LSSI maps of the region of Murcia (Spain) where land subsidence occurred in the past due to groundwater overdraft. A LSSI map was initially generated with known FR. Then, additional Conditioning Factors (CFs) with increased spatial resolution were used to train several ML models and generate a new LSSI map. The Extra-Trees Classifier (ETC) outperformed the other approaches, achieving the best performance with a weighted average precision and F1-Score of 0.96, after optimizing its hyperparameters. Then, a third LSSI map was calculated using the FR method and observations of land subsidence from InSAR (Interferometric Synthetic Aperture Radar). This study shows that the effectiveness of using several CFs depends on the added information of each layer. Moreover, the comparison between the different LSSI maps and InSAR data highlights the crucial role of the spatial resolution for accurate mapping, thus enhancing land subsidence risk assessment

Discovery of Boronic Acids-Based β-Lactamase Inhibitors Through In Situ Click Chemistry

In this study, we evaluated in situ click chemistry as a platform for discovering boronic acid-based beta-lactamase inhibitors (BLIs). Unlike conventional drug discovery approaches requiring multi-step synthesis, protection strategies, and extensive screening, the in situ method can allow for the generation and identification of potent beta-lactamase inhibitors in a rapid, economic, and efficient way. Using KPC-2 (class A carbapenemase) and AmpC (class C cephalosporinase) as templates, we demonstrated their ability to catalyse azide-alkyne cycloaddition, facilitating the formation of triazole-based beta-lactamase inhibitors. Initial screening of various beta-lactamases and boronic warheads identified compound 3 (3-azidomethylphenyl boronic acid) as the most effective scaffold for kinetic target-guided synthesis (KTGS). KTGS experiments with AmpC and KPC-2 yielded triazole inhibitors with Ki values as low as 140 nM (compound 10a, AmpC) and 730 nM (compound 5, KPC-2). Competitive inhibition studies confirmed triazole formation within the active site, while an LC-MS analysis verified that the reversible covalent interaction of boronic acids did not affect detection of the in situ-synthesised product. While KTGS successfully identified potent inhibitors, limitations in amplification coefficients and spatial constraints highlight the need for optimised warhead designs. This study validates KTGS as a promising strategy for BLI discovery and provides insights for further refinement in fighting beta-lactamase-mediated antibiotic resistance

Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major β-lactamases

The N-terminal sequences of the two major β-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca β-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded β-lactamase of Citrobacter diversus belong to class A

The X-Linked Inhibitor of Apoptosis Protein Inhibitor Embelin Suppresses Inflammation and Bone Erosion in Collagen Antibody Induced Arthritis Mice

Copyright © 2015 Anak A. S. S. K. Dharmapatni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Objective. To investigate the effect of Embelin, an inhibitor of X-Linked Inhibitor of Apoptosis Protein (XIAP), on inflammation and bone erosion in a collagen antibody induced arthritis (CAIA) in mice. Methods. Four groups of mice ( per group) were allocated: CAIA untreated mice, CAIA treated with Prednisolone (10 mg/kg/day), CAIA treated with low dose Embelin (30 mg/kg/day), and CAIA treated with high dose Embelin (50 mg/kg/day). Joint inflammation was evaluated using clinical paw score and histological assessments. Bone erosion was assessed using micro-CT, tartrate resistant acid phosphatase (TRAP) staining, and serum carboxy-terminal collagen crosslinks (CTX-1) ELISA. Immunohistochemistry was used to detect XIAP protein. TUNEL was performed to identify apoptotic cells. Results. Low dose, but not high dose Embelin, suppressed inflammation as reflected by lower paw scores () and lower histological scores for inflammation. Low dose Embelin reduced serum CTX-1 () and demonstrated lower histological score and TRAP counting, and slightly higher bone volume as compared to CAIA untreated mice. XIAP expression was not reduced but TUNEL positive cells were more abundant in Embelin treated CAIA mice. Conclusion. Low dose Embelin suppressed inflammation and serum CTX-1 in CAIA mice, indicating a potential use for Embelin to treat pathological bone loss

Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

<p>Abstract</p> <p>Background</p> <p>Plasmid encoded ^<it>bla</it>CTX-M enzymes represent an important sub-group of class A β-lactamases causing the ESBL phenotype which is increasingly found in <it>Enterobacteriaceae </it>including <it>Klebsiella </it>spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.</p> <p>Methods</p> <p>Multiple displacement amplified DNA derived from 20 <it>K. pneumoniae </it>and 34 <it>K. oxytoca </it>clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.</p> <p>Results</p> <p>Nine out of 20 <it>K. pneumoniae </it>clinical isolates had a ^<it>bla</it>CTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 <it>K. oxytoca </it>clinical isolates. Molecular identification and differentiation between ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. <it>In silico </it>analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located ^<it>bla</it>OXY and K1-genes in <it>Klebsiella </it>spp. and K1-like genes in other <it>Enterobacteriaceae</it>.</p> <p>Conclusion</p> <p>The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of ^<it>bla</it>CTX-M, and ^<it>bla</it>OXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.</p

Eight basic principles for the elaboration of public policies and development projects for the Pantanal.

Abstract: The Pantanal is considered the largest continuous freshwater wetland in the world, and its sustainable use requires a unified conceptual framework. The lengthy process to establish public policies has contributed to the increasing vulnerability of the Pantanal. Given the need for a conceptual basis to help this process, we elaborate a list of eight basic principles based on the accumulated scientific evidence: (i) Consider the Paraguay River Basin a management unity; (ii) Establish rules that follow the concept of restricted use approach; (iii) Ensure the ecologically sustainable use of the Pantanal; (iv) Maintain the environmental heterogeneity and functionality in the Pantanal landscapes; (v) Maintain the hydrological integrity and connectivity; (vi) Ensure the environmental representativeness of the protected areas network; (vii) Provide economic incentives for conservationist use of the land; and (viii) Recognize and protect traditional people, their values, resources, and way of living. However, the elaboration of public policies should be a participatory and inclusive decision‐making process towards a more just and sustainable future.Online first