OBJECTIVES: The excessive use of antibiotics has driven β-lactam resistance in Escherichia coli, with the two-component system (TCS) playing a key role in regulating virulence and resistance genes. The EnvZ/OmpR TCS, inv...OBJECTIVES: The excessive use of antibiotics has driven β-lactam resistance in Escherichia coli, with the two-component system (TCS) playing a key role in regulating virulence and resistance genes. The EnvZ/OmpR TCS, involving the histidine kinase EnvZ and regulator OmpR, influences porin genes ompC and ompF. The authors' previous work suggested that EnvZ may regulate β-lactam antibiotic resistance, but the mechanism remains unclear. This study aimed to explore how EnvZ regulates β-lactam antibiotic resistance and virulence mechanisms in E. coli. METHODS: An envZ deletion mutant (ΔenvZ) was constructed from an imipenem-resistant E. coli strain (Sx181-128). Phenotypic assays evaluated antibiotic susceptibility, environmental tolerance, adhesion and motility. RNA sequencing (RNA-seq) compared transcriptional profiles between Sx181-128 and ΔenvZ. RESULTS: The envZ deletion mutant exhibited a 2-fold increase in susceptibility to six β-lactam antibiotics (cefoxitin, meropenem, imipenem, cefepime, amoxicillin and aztreonam) compared with the resistant strain Sx181-128, along with compromised tolerance especially under hypertonic condition. RNA-seq analysis revealed 338 differentially expressed genes (116 up-regulated, 222 down-regulated), primarily associated with porins, transporters, amino sugar and nucleotide sugar metabolism and flagellar assembly. Mechanistically, envZ deletion increased membrane permeability by dysregulating ompC and ompF expression, enhancing β-lactam antibiotic uptake. Additionally, ΔenvZ displayed increased adhesion to HeLa cells, bacterial motility and biofilm-forming capacity, suggesting a dual role for EnvZ in modulating both antibiotic resistance and virulence. CONCLUSIONS: EnvZ regulates β-lactam resistance by modulating porin expression and membrane permeability, while also influencing virulence traits like adhesion and motility. These findings highlight TCS-mediated resistance mechanisms and offer potential targets for novel antimicrobials or vaccines against E. coli.
Polymyxins serve as a 'last-line' defence against Gram-negative bacterial infections and are frequently used in critically ill patients with multidrug-resistant pathogens. However, polymyxin-associated acute kidney injur...Polymyxins serve as a 'last-line' defence against Gram-negative bacterial infections and are frequently used in critically ill patients with multidrug-resistant pathogens. However, polymyxin-associated acute kidney injury (PA-AKI) remains a major factor limiting their clinical application. This review examines the pathophysiology, risk factors and clinical management of PA-AKI, providing updated perspectives on its prevention and treatment. Reducing exposure to concomitant nephrotoxic agents, together with timely and standardized monitoring of serum creatinine, urine volume and polymyxin concentrations, plays a key role in mitigating the risk and progression of PA-AKI. Although several biomarkers show promise for the early prediction of PA-AKI and may enable earlier intervention, many have not yet undergone extensive clinical validation. There is a clear need to incorporate real-world evidence into clinical practice guidelines for polymyxin use. Further research should focus on identifying genetic risk factors for PA-AKI and developing novel polymyxin analogues with reduced nephrotoxicity.
Olivares-Navarro P, Pérez-Rodríguez MT, Sousa A
… +22 more, Goikoetxea-Agirre AJ, Blanco Vidal MJ, Plata A, León E, Buzón-Martín L, Pulido-Navazo Á, Boix-Palop L, Retamar-Gentil P, Armiñanzas-Castillo C, Fernández-Natal I, Del Arco Jiménez A, Jover-Sáenz A, Fernández-Suárez J, Martín-Aspas A, Smithson-Amat A, Bahamonde-Carrasco A, Natera-Kindelán C, Martínez Pérez-Crespo PM, López-Hernández I, Rodríguez-Baño J, López-Cortés LE, PROBAC/GEIRAS-SEIMC/SAMICEI
J Antimicrob Chemother
· 2026 Apr · PMID 41953964
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OBJECTIVE: The incidence of bloodstream infection (BSIs) due to extended-spectrum β-lactamase (ESBL) producing Escherichia coli is increasing worldwide. There is controversy as to whether ESBL production in itself is ass...OBJECTIVE: The incidence of bloodstream infection (BSIs) due to extended-spectrum β-lactamase (ESBL) producing Escherichia coli is increasing worldwide. There is controversy as to whether ESBL production in itself is associated with higher mortality. The aim of this study is to evaluate the impact of ESBL production on mortality in BSIs due to E. coli considering the effect of confounders. METHODS: PROBAC study is a prospective, multicentre, cohort study performed in 26 Spanish hospitals (October 2016-March 2017). All patients with E. coli BSIs were included. The outcome variable was all-cause 30-day mortality. Confounding was controlled by calculating a propensity score (PS) for ESBL production using baseline variables. PS were used as covariable, for matching, for inverse probability of treatment weight analysis and for stratified analysis within the PS quartiles. RESULTS: A total of 2394 cases were included, of which 322 (13.5%) were ESBL-producing isolates. The frequency of appropriate empirical treatment, in ESBL-producing and non-ESBL-producing isolates, was 53.7% and 92.0%, respectively. Thirty-day mortality was 14.6% in ESBL-producing isolates versus 9.6% in non-ESBL-producing isolates (P = 0.006), for a crude OR of 1.61 (95% CI: 1.14-2.27; P = 0.006). When we adjusted by PS and appropriate empirical treatment, the OR changed to 1.12 (95% CI: 0.75-1.67; P = 0.584). Other PS applications provided similar results. CONCLUSION: BSIs due to ESBL-producing E. coli were associated with higher mortality in the crude analyses; however, the estimate of the association is reduced after adjustment for baseline variables and empirical therapy, and is not significant in matched analysis.
Luck ME, Martin A, Punja S
… +4 more, Kamar J, Zuchinali P, Edgecomb AG, Ellis JJ
J Antimicrob Chemother
· 2026 Apr · PMID 41953963
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BACKGROUND: Uncomplicated urinary tract infections (UTIs) affect ∼50%-60% of women. Treatment failure can have adverse effects on antibiotic resistance, healthcare utilization and quality of life. The lack of consistentl...BACKGROUND: Uncomplicated urinary tract infections (UTIs) affect ∼50%-60% of women. Treatment failure can have adverse effects on antibiotic resistance, healthcare utilization and quality of life. The lack of consistently applied definitions of treatment failure prevents comparisons between studies of UTI treatments. OBJECTIVES: We conducted a systematic literature review to investigate definitions of treatment failure in UTI and the corresponding failure rates. METHODS: MEDLINE, Embase and CENTRAL databases were searched from 2011 to 2024 and relevant conference proceedings from 2021 to 2024 for English language studies reporting rates of treatment failure in females aged ≥12 years with uncomplicated UTI. RESULTS: Publications included reported 14 clinical trials, 11 non-interventional observational studies with chart review and 10 healthcare database studies. Treatment failure definitions were classified as microbiological, clinical, antibiotic prescription based or a composite of these. Evaluation timepoints typically ranged from 1 to 30 days post-treatment. In clinical trials, failure rates varied from 0.8% to 83%, often with marked differences between microbiological, clinical and prescription definitions within the same trial. Rates of treatment failure using combined endpoints, including prescription failure with a healthcare encounter in database studies, were generally more consistent (6.87%-16.7%). Few studies assessed time to symptom resolution. Prescription failure or additional healthcare visits frequently occurred after a median 2-4 weeks. CONCLUSIONS: Treatment failure definitions have been variably defined in the literature. Symptom scores in clinical trials and the need for additional antibiotics or healthcare visits are meaningful outcomes that could underly treatment failure definitions in future studies. The optimal time for each outcome assessment needs further evaluation.
OBJECTIVES: Cefiderocol is an important treatment option for infections caused by multidrug-resistant Gram-negative bacteria; however, resistance among New Delhi metallo-β-lactamase-producing Enterobacterales (NDME) is i...OBJECTIVES: Cefiderocol is an important treatment option for infections caused by multidrug-resistant Gram-negative bacteria; however, resistance among New Delhi metallo-β-lactamase-producing Enterobacterales (NDME) is increasingly reported, leaving limited therapeutic alternatives. This study evaluated the in vitro activity of the minocycline-biapenem combination against cefiderocol-resistant NDME. METHODS: Twenty-three non-duplicate clinical NDME isolates with cefiderocol MIC ≥ 16 mg/L determined by reference broth microdilution in iron-depleted cation-adjusted Mueller-Hinton broth were analysed. Checkerboard assays were performed to assess combination activity using fractional inhibitory concentration indices (FICIs), sub-MIC interaction analysis and cumulative inhibition heatmaps. Time-kill assays were conducted using three representative isolates producing NDM-1 or NDM-5. RESULTS: Susceptibility rates for minocycline and biapenem tested alone were 52% and 13%, respectively. The minocycline-biapenem combination demonstrated synergistic activity (FICI ≤ 0.5) in 22 of 23 isolates (96%), with a median FICI of 0.063. Synergy was consistently observed across Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae. Sub-MIC analyses revealed asymmetric pharmacodynamic interactions, with sustained reductions in biapenem MIC at minocycline exposures as low as 1/512× baseline MIC. Cumulative inhibition heatmaps showed growth inhibition in 100% of isolates at the susceptible breakpoint concentrations of minocycline (4 mg/L) and biapenem (1 mg/L). Time-kill assays confirmed rapid and exposure-dependent bactericidal activity at clinically achievable concentration pairs. CONCLUSIONS: The minocycline-biapenem combination showed consistent in vitro synergistic and bactericidal activity against cefiderocol-resistant NDME across multiple species and NDM variants. These findings support further pharmacodynamic and translational evaluation of this combination as a potential therapeutic option for cefiderocol-refractory infections.
Ferrara M, Maccario V, Barrera F
… +11 more, Ponzetta L, Di Girolamo L, Arrue Diaz D, Bo G, Orofino GC, Maiese D, Soloperto S, De Nicolò A, D'Avolio A, Calcagno A, Bonora S
OBJECTIVES: Long-acting (LA) intramuscular cabotegravir (CAB) and rilpivirine (RPV) represent a novel antiretroviral therapy (ART) option for people with HIV (PWH), offering improved adherence and patient convenience. Th...OBJECTIVES: Long-acting (LA) intramuscular cabotegravir (CAB) and rilpivirine (RPV) represent a novel antiretroviral therapy (ART) option for people with HIV (PWH), offering improved adherence and patient convenience. The aim was to evaluate plasma and intracellular (IC) exposure of CAB and RPV in PWH switching from oral ART to LA injectable regimens and to assess correlations with virological outcomes and clinical parameters. METHODS: This is a monocentric prospective observational cohort study. We enrolled 177 PWH who switched to CAB/RPV intramuscular injections every 8 weeks without oral lead-in. Plasma and IC drug concentrations in peripheral blood mononuclear cells were measured at baseline and at every follow-up visit over 12 months. Virological suppression, immunological parameters and correlations with PK data were analysed. RESULTS: At baseline, 93.6% of participants had undetectable viral load (<20 copies/mL). CAB and RPV plasma trough concentrations showed moderate intra-individual and high inter-individual variability, with RPV IC accumulation ∼ 5-fold higher than plasma, and CAB IC exposure about 15% of plasma levels. A transient decrease in plasma and IC concentrations was observed at month 3, without impact on virological suppression. Virological blips occurred in 15.3% of participants, with no significant association to drug concentrations. Two virological failures are reported with onset of NNRTI and INSTI major and accessory resistance mutation. CONCLUSIONS: LA CAB and RPV achieve plasma exposures consistent with clinical trials, maintaining virological suppression despite pharmacokinetic variability. Intracellular accumulation, especially of RPV, may contribute to sustained virological control. These findings support the use of LA CAB/RPV as a robust ART option in real-world clinical practice.
Al-Mir H, Jeyakumar PZ, Kostyanev T
… +18 more, Khan FA, Rasmussen LB, Emmad M, Abegaz FA, Teixeira Dos Santos P, Kwon SY, Prathan R, Luangtongkum T, Chanchaithong P, Santanirand P, Kamjumpho W, Mogeni OD, Guarnacci T, Holm M, Paveenkittiporn W, Poudyal N, Chuanchuen R, Hendriksen RS
J Antimicrob Chemother
· 2026 Apr · PMID 41934336
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OBJECTIVES: The increasing prevalence of antimicrobial resistance (AMR) in Escherichia coli, notably those producing AmpC, ESBL and carbapenemases among food-producing animals, poses a significant public health threat, e...OBJECTIVES: The increasing prevalence of antimicrobial resistance (AMR) in Escherichia coli, notably those producing AmpC, ESBL and carbapenemases among food-producing animals, poses a significant public health threat, especially in low- and middle-income countries. With support from the Fleming Fund and in alignment with the WHO, FAO and WOAH tripartite Tricycle project, the EQASIA Matrix External Quality Assessment (EQA) scheme aimed at evaluating laboratories' proficiency from different One Health (OH) sectors in detecting resistant E. coli from food-like matrices in South and South-East Asia. METHODS: Between 2021 and 2024, four Matrix EQA rounds were implemented across OH sectors. Each round involved four lyophilized simulated meat samples spiked with well-characterized E. coli strains expressing β-lactam resistance phenotypes. Participating laboratories performed selective isolation, species identification, antimicrobial susceptibility testing (AST) and phenotypic classification using routine methods. Results were submitted via a secure online platform and evaluated against expected outcomes, using CLSI-based interpretive criteria and a standardized error categorization system. RESULTS: Twenty-one laboratories from 10 countries participated across the four rounds. Accuracy in E. coli identification declined from 75% in 2021 to 23.1% in 2024. Only one laboratory achieved full identification accuracy in any round. AST performance showed inter-laboratory variation, with deviation rates ranging from 0.5% to 41%. Misclassification of resistance phenotypes, particularly AmpC and combined ESBL + AmpC, was common. Quality control testing improved modestly over time but remained incomplete. CONCLUSIONS: The Matrix EQA revealed critical insights into regional laboratory capacity and highlighted persistent technical gaps in detecting complex resistance mechanisms from food matrices. With the conclusion of the Fleming Fund, maintaining these gains will require sustained national investment, EQA integration into AMR strategies and continued regional coordination to support Tricycle-aligned surveillance.
Law D, Ayrton J, Kennedy A
… +1 more, Cornelissen K
J Antimicrob Chemother
· 2026 Apr · PMID 41934335
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OBJECTIVES: To evaluate the penetration of olorofim, a novel antifungal agent into the tissues of the rat. MATERIALS AND METHODS: Quantitative whole-body autoradiography was used to assess the distribution and tissue pen...OBJECTIVES: To evaluate the penetration of olorofim, a novel antifungal agent into the tissues of the rat. MATERIALS AND METHODS: Quantitative whole-body autoradiography was used to assess the distribution and tissue penetration in male and female rats following a single 2-hour IV infusion of [14C]-olorofim. RESULTS: Radioactivity was rapidly and extensively distributed throughout many tissues in both sexes with maximal concentrations observed soon after start of the infusion. Thereafter radioactivity declined and at 336 h (final timepoint), concentrations of radioactivity in most tissues were below the limit of quantification. The pattern of radioactive distribution was similar at all timepoints with concentrations greatest in liver, kidney cortex, adrenal cortex, abdominal and brown fat; the lowest concentrations were observed in the whole eye and at the bone surface (with levels similar to plasma in bone marrow and uveal tract/retina). Tissue:plasma radioactivity concentration ratios for most tissues were above unity during the period 2.25 to 72 h post-start of infusion. Radioactivity was detected in tissues frequently reported as sites of systemic fungal infections such as the lung, brain, kidney, bone and eye. Pharmacokinetic analyses showed that at early timepoints >75% of circulating radiolabelled material was intact olorofim confirming that olorofim is present in these key tissues at levels similar to or exceeding those seen in plasma. CONCLUSIONS: Olorofim distributes widely into rat tissues including those frequently infected by fungi, indicating its potential to treat systemic fungal infections caused by susceptible species, a finding in agreement with recent clinical reporting from a Phase 2 trial.
Beijer G, Wallander K, Söderquist B
… +4 more, Giske CG, Breuer O, Eriksen J, Eliasson E
J Antimicrob Chemother
· 2026 Apr · PMID 41934334
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OBJECTIVES: To characterize the population pharmacokinetics of unbound cloxacillin in patients undergoing total arthroplasty of the hip (THA) or knee (TKA), and to explore alternative dosing regimens for cloxacillin prop...OBJECTIVES: To characterize the population pharmacokinetics of unbound cloxacillin in patients undergoing total arthroplasty of the hip (THA) or knee (TKA), and to explore alternative dosing regimens for cloxacillin prophylaxis. METHODS: Plasma concentrations of total and unbound cloxacillin from 200 patients undergoing primary elective THA (n & 95) or TKA (n & 105) were analysed. Intravenous cloxacillin doses of 2 g were administered pre-surgery, and repeated after 2 and 6 hours. Samples (n & 496) were analysed using HPLC-MS/MS. Non-linear mixed-effects modelling was performed to develop a population pharmacokinetic model describing unbound cloxacillin exposure. Using this model, alternative prophylaxis regimens were explored with Monte Carlo simulations. RESULTS: A two-compartment model with non-linear protein binding adequately described the data. Estimated glomerular filtration rate (eGFR) and body weight (kg) were significant covariates on unbound cloxacillin clearance. In 13% of patients sampled at the end of surgery (n & 25/187), unbound cloxacillin <2 mg/L was observed. A model-predicted 18-22% (n & 36-43/200) of patients failed to sustain plasma levels ≥2 mg/L throughout the two-hour dosing interval with the current regimen. In contrast, a continuous 1 g/h infusion after a 1 g loading dose would ensure target attainment in >99% of patients, according to the model predictions. CONCLUSIONS: For many THA and TKA patients, the current cloxacillin prophylaxis regimen may fail to provide adequate target site concentrations during the entire surgical procedure. Transitioning to a prolonged infusion protocol could increase attainment of PK/PD targets without exceeding the currently recommended total perioperative dose amounts.
BACKGROUND: The emergence and spread of hypervirulent Klebsiella pneumoniae (hvKp) poses a serious and growing challenge to public health worldwide. However, the knowledge regarding hvKp of animal origin remains very lim...BACKGROUND: The emergence and spread of hypervirulent Klebsiella pneumoniae (hvKp) poses a serious and growing challenge to public health worldwide. However, the knowledge regarding hvKp of animal origin remains very limited. In this study, we characterized a colistin-resistant hvKp isolate obtained from healthy chicken faeces and gained insight into the molecular basis of hypervirulence and colistin resistance. METHODS: Antimicrobial susceptibility testing was conducted by broth microdilution. The hypermucoviscous phenotype was determined by string tests. Whole-genome sequencing (WGS) was performed using a combination of Illumina NovaSeq/Oxford Nanopore PromethION platforms. Sequence alignment and complement assays were used to identify the mutations conferring colistin resistance. The virulence was investigated using both Galleria mellonella larvae and mice infection models. RESULTS: This strain KP20 displayed a hypermucoviscous phenotype and strong biofilm-forming capacity. WGS revealed that strain KP20 carried a pLVPK-like virulence plasmid with a novel replicon profile (IncFIB/RepB), harbouring key hypervirulence-associated genes, including iucABCD-iutA, iroBCDN, peg-344 and rmpA/rmpA2. Moreover, a plasmid harbouring the trimethoprim resistance gene dfrA50, pKP20-2, was identified as a member of the phage-like plasmids: genetic elements that function both as phages and plasmids. Sequence analysis and functional confirmation suggested that mutations in the crrB gene contributed to the colistin resistance observed in this strain. The virulence of KP20 was confirmed in the animal infection models. CONCLUSIONS: In this study, a high-risk ST412-K57 colistin-resistant hvKp of animal origin was identified and characterized. According to the One Health concept, our findings highlight the critical need for implementing enhanced molecular surveillance of hvKp in animals.
Maertens J, Chen SCA, Thompson GR
… +11 more, Donovan F, Dane A, Birch M, Pinder C, Bennett J, Ravenna V, Marin Fernandez O, Ross G, Zinzi D, Rex JH, Phase 2b Olorofim Salvage Therapy Study Group
J Antimicrob Chemother
· 2026 Mar · PMID 41914555
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BACKGROUND: Olorofim is an antifungal agent with a novel mechanism of action against Aspergillus spp., rare moulds and dimorphic fungi. Preclinical studies reported unidirectional antagonism between olorofim and mould-ac...BACKGROUND: Olorofim is an antifungal agent with a novel mechanism of action against Aspergillus spp., rare moulds and dimorphic fungi. Preclinical studies reported unidirectional antagonism between olorofim and mould-active azoles (e.g. azoles other than fluconazole) against Aspergillus fumigatus in which the azole reduces the antifungal effect of olorofim. METHODS: To assess the potential clinical impact of this preclinical finding, we compared outcomes of olorofim with and without concomitant azoles from a Phase 2b salvage study in patients with invasive fungal diseases with few or no treatment options. RESULTS: Analyses are limited by the study size but outcomes were similar for olorofim with and without an azole in combination (whether mould-active or not) at Days 42 (the primary study endpoint) and 84 for both Mycoses Study Group-European Organization for Research and Treatment of Cancer (MSG-EORTC) responses and for all-cause mortality in patients with aspergillosis (77 not receiving an azole, 24 receiving an azole in combination), in patients with coccidioidomycosis (11 not receiving an azole, 30 receiving an azole in combination) and in patients with other fungal infections (50 not receiving an azole, 10 receiving an azole in combination). In addition, the rate of drug-induced liver injury was not increased in patients receiving combination with an azole. CONCLUSIONS: Although limited by small numbers of patients for individual fungi, this analysis did not demonstrate a pattern of reduced clinical efficacy or tolerability of olorofim when given in combination with either the mould-active azoles or with fluconazole.Trial registration number. NCT03583164.
Baiardi G, Pontali E, Marini V
… +3 more, Cristina ML, Sartini M, Mattioli F
J Antimicrob Chemother
· 2026 Mar · PMID 41914554
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BACKGROUND AND OBJECTIVES: Oritavancin is a lipoglycopeptide with sustained bactericidal activity against Gram-positive bacteria due to its prolonged half-life. This Systematic Review aimed to extrapolate, from in vitro/...BACKGROUND AND OBJECTIVES: Oritavancin is a lipoglycopeptide with sustained bactericidal activity against Gram-positive bacteria due to its prolonged half-life. This Systematic Review aimed to extrapolate, from in vitro/in vivo or clinically study, the most relevant PK/PD target to inform therapeutic drug monitoring-guided oritavancin dose optimization in clinical practice. MATERIALS AND METHODS: Following the PRISMA 2020 Statement and adopting the PICO strategy, a comprehensive search was conducted in PubMed, Scopus and Cochrane databases up to September 2025. RESULTS: Of 186 articles screened, 52 were considered eligible for full-text assessment. Nine studies were included and proceeded with data extraction and synthesis steps. In vitro studies showed a marked concentration-dependent bactericidal activity at fCmax > 4-16 mg/L against different bacterial strains, further confirmed by in vivo animal models (fCmax/MIC > 6 to 14). However, the only identified in-human daily repeated doses study supported the findings of an exposure-response relationship with %fT > MIC as predictive of microbiological and clinical success. CONCLUSIONS: The peculiar pharmacokinetics profile of oritavancin results in a borderline collinearity between the two PK/PD indices fCmax/MIC and %fT > MIC in relation to microbiological and clinical success rates. On the basis of available in vitro/in vivo data supporting concentration-dependent killing activity, a single 1200 mg oritavancin dose should be adequate for most infections. In special patient populations, or when multidose oritavancin regimens are adopted for long-term antibiotic treatment, therapeutic drug monitoring supported by expert clinical pharmacological advice may be valuable to optimize the initial and next-dose strategy (1200 mg or 800 mg) and to define the timing of re-administration.
J Antimicrob Chemother
· 2026 Mar · PMID 41913954
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BACKGROUND AND OBJECTIVES: Metallo-β-lactamase (MBL)-producing Enterobacterales represent a major public health threat because they confer resistance to all β-lactams except monobactams. The most prevalent MBLs include V...BACKGROUND AND OBJECTIVES: Metallo-β-lactamase (MBL)-producing Enterobacterales represent a major public health threat because they confer resistance to all β-lactams except monobactams. The most prevalent MBLs include VIM, IMP, and NDM types, which render all clinically approved β-lactamase inhibitors ineffective. This study evaluates whether cyanide-containing molecules such as the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) or the cytochrome C oxidase inhibitor potassium cyanide (KCN) can restore carbapenem susceptibility in MBL-producing strains by disrupting zinc-dependent enzymatic activity. METHODS: A series of isogenic Escherichia coli TOP10 and MG1655 strains carrying the shuttle vector pUCP24 harbouring various MBLs and clinical strains were included in this study. The strains were subjected to antimicrobial susceptibility testing by broth microdilution, checkerboard assays, determination of the 50% inhibitory concentration (IC50) of carbapenemase activity, kinetic assays and time-kill curve assays. RESULTS: CCCP, FCCP and KCN restored carbapenem (imipenem) susceptibility in E. coli strains producing VIM-1-like enzymes. Synergy was observed in checkerboard assays, and reduction of bacterial growth was confirmed by time-kill experiments. The addition of Zn2+ reversed this effect, supporting a Zn2+-chelating or Zn2+-displacing mechanism. Non-VIM-1-like MBLs, including VIM-2, IMP-1 and NDM-1, were not affected by the addition of cyanide-containing compounds, indicating strong selectivity of these inhibitory effects. IC50 measurements confirmed the selective activity of CCCP, FCCP and KCN against VIM-1-like MBLs. CONCLUSIONS: CCCP, FCCP and KCN exhibited inhibitory activity against VIM-1-like MBLs, restoring imipenem susceptibility in E. coli at very low concentrations (0.04-19.55 µM). Thus, certain efflux pump inhibitors, as well as the gaseous biological mediator cyanide, have a novel action as inhibitors of MBL enzymatic activity, which, in turn, renders them potentially useful as adjunct antibacterial agents.
Hendrickx S, Ilbeigi K, Thoré ESJ
… +17 more, Bertram MG, Calvo-Alvarez E, Cintesun S, Olías-Molero AI, Corral MJ, Mateo-Barrientos M, Estaquier J, Pomel S, Alunda JM, Gul S, Van Bocxlaer K, Frézard F, Tavares J, Cordeiro Da Silva A, Costi MP, Maes L, Caljon G
J Antimicrob Chemother
· 2026 Mar · PMID 41913953
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Given the medical importance and challenges related to kinetoplastid diseases, a strategic roadmap is needed for the identification of high-quality leads and drug development candidates. Within the aim to deliver more co...Given the medical importance and challenges related to kinetoplastid diseases, a strategic roadmap is needed for the identification of high-quality leads and drug development candidates. Within the aim to deliver more compelling proof-of-concept read-outs, this part proposes a systematic flow-chart of laboratory experiments and decision criteria, focusing on African trypanosomiasis, Chagas disease and visceral and cutaneous leishmaniasis. Next to precision experimental design and reporting, an overview is provided of various complementary laboratory models reproducing kinetoplastid infection and disease. Technical aspects of conventional in vitro and in vivo approaches and, more recently, in silico methods are presented with reference to specific preclinical R&D stages from 'hit finding' to 'profiling of a confirmed hit', covering the expertise areas of medicinal chemistry, primary pharmacology, (eco)toxicology, pharmacokinetics and pharmaceutics (Figure 1).
OBJECTIVES: Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted by Gram-negative bacteria that have emerged as important mediators of antibiotic resistance dissemination. This study aimed to elucidate t...OBJECTIVES: Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted by Gram-negative bacteria that have emerged as important mediators of antibiotic resistance dissemination. This study aimed to elucidate the structural, functional and proteomic characteristics of OMVs derived from Escherichia coli carrying the blaNDM-5 gene and to determine their contribution to carbapenem resistance transfer and bacterial adaptation. METHODS: OMVs were isolated from E. coli strains with or without blaNDM-5 expression and characterized by transmission electron microscopy, dynamic light scattering and zeta potential analysis. The presence of blaNDM-5 and β-lactamase activity in OMVs was confirmed by PCR and ELISA. Horizontal gene transfer was evaluated using a bioluminescent E. coli recipient strain under selective pressure. LC-MS/MS proteomics was performed to assess changes in OMV protein composition associated with blaNDM-5 expression. RESULTS: OMVs from blaNDM-5-positive E. coli encapsulated both blaNDM-5-bearing plasmids and catalytically active NDM-5 carbapenemase, enabling horizontal transfer of functional resistance to susceptible recipients. Acquisition of OMV-delivered plasmids increased meropenem MICs by over 500-fold, while OMV-associated β-lactamase activity reduced antibiotic efficacy in the extracellular environment and protected nearby susceptible bacteria. Proteomic profiling further revealed that blaNDM-5 expression was accompanied by broad changes in OMV protein composition, consistent with global cellular adaptations to carbapenem exposure. CONCLUSIONS: bla NDM-5-positive OMVs promote carbapenem resistance through dual mechanisms involving plasmid-mediated gene transfer and extracellular antibiotic degradation. These findings extend prior work on OMV-associated carbapenemase activity and identify bacterial vesicles as an underappreciated but potentially important contributor to the dissemination and maintenance of carbapenem resistance.
INTRODUCTION: Aligning national standard treatment guidelines (STGs) with WHO AWaRe Book recommendations where appropriate is a key antimicrobial stewardship goal. Antibiotic recommendations in national STGs vary signifi...INTRODUCTION: Aligning national standard treatment guidelines (STGs) with WHO AWaRe Book recommendations where appropriate is a key antimicrobial stewardship goal. Antibiotic recommendations in national STGs vary significantly, creating gaps that require evaluation to help address rising AMR rates. METHODS: We analysed adult STGs from 24 countries in six WHO regions for 11 infections commonly seen across countries in the primary care section of the WHO AWaRe Book. These included acute otitis media (AOM), pharyngitis, sinusitis, community-acquired pneumonia (CAP), chronic obstructive pulmonary disease, lower urinary tract infections, gastroenteritis (bloody and non-bloody), oral and dental infections, skin and soft tissue infections and enteric fever. National STGs were compared with WHO AWaRe recommendations to assess alignment of first-line antibiotic recommendations. RESULTS: 522 different first-line oral antibiotic regimens were collected from the different STGs in 24 countries across 6 WHO regions. Access antibiotics were recommended in 65.7% of the regimens, while Watch antibiotics accounted for 34.3% of first-line recommendations globally, with limited recommendations of Reserve antibiotics. The Western Pacific Region (76.4%), showed the highest proportions of Access group antibiotic recommendations. Infection-specific analysis showed significant variation in alignment, with a higher alignment for enteric fever, non-bloody gastroenteritis and cystitis, and a lower alignment for CAP, oral and dental infections, and AOM (P = 0.0089). CONCLUSION: First-line antibiotic recommendations in adult STGs showed moderate alignment with WHO AWaRe Book treatment guidance, with regional variability. Aligning first-line recommendations with the WHO AWaRe Book guidance in primary care would assist stewardship programmes.