Transferable AmpCs in Klebsiella pneumoniae: interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications.

Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.

In vitro dynamics and mechanisms of cefiderocol resistance development in wild-type, mutator and XDR Pseudomonas aeruginosa.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to cefiderocol in Pseudomonas aeruginosa. METHODS: Cefiderocol resistance evolution was analysed in WT PAO1, PAOMS (mutS mutator derivate) and three XDR clinical isolates belonging to ST111, ST175 and ST235 clones. Strains were incubated in triplicate experiments for 24 h in iron-depleted CAMHB with 0.06-128 mg/L cefiderocol. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 128 mg/L for 7 consecutive days. Two colonies per strain and experiment were characterized by determining the susceptibility profiles and WGS. RESULTS: Evolution of resistance was significantly enhanced in PAOMS, but was variable for the XDR strains, including levels similar to PAOMS (ST235), similar to PAO1 (ST175) or even below PAO1 (ST111). WGS revealed 2-5 mutations for PAO1 lineages and 35-58 for PAOMS. The number of mutations in the XDR clinical strains ranged from 2 to 4 except for one of the ST235 experiments in which a mutL lineage was selected, thus increasing the number of mutations. The most frequently mutated genes were piuC, fptA and pirR, related to iron uptake. Additionally, an L320P AmpC mutation was selected in multiple lineages and cloning confirmed its major impact on cefiderocol (but not ceftolozane/tazobactam or ceftazidime/avibactam) resistance. Mutations in CpxS and PBP3 were also documented. CONCLUSIONS: This work deciphers the potential resistance mechanisms that may emerge upon the introduction of cefiderocol into clinical practice, and highlights that the risk of resistance development might be strain-specific even for XDR high-risk clones.

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis.

Background: Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods: A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings: A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum ß-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation: While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance. Funding: MSD and the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.

Blood-brain barrier dysfunction underlying Alzheimer's disease is induced by an SSAO/VAP-1-dependent cerebrovascular activation with enhanced Aß deposition.

Dysfunctions of the vascular system directly contribute to the onset and progression of Alzheimer's disease (AD). The blood-brain barrier (BBB) shows signs of malfunction at early stages of the disease. When Abeta peptide (Aß) is deposited on brain vessels, it induces vascular degeneration by producing reactive oxygen species and promoting inflammation. These molecular processes are also related to an excessive SSAO/VAP-1 (semicarbazide-sensitive amine oxidase) enzymatic activity, observed in plasma and in cerebrovascular tissue of AD patients. We studied the contribution of vascular SSAO/VAP-1 to the BBB dysfunction in AD using in vitro BBB models. Our results show that SSAO/VAP-1 expression is associated to endothelial activation by altering the release of pro-inflammatory and pro-angiogenic angioneurins, most highly IL-6, IL-8 and VEGF. It is also related to a BBB structure alteration, with a decrease in tight-junction proteins such as zona occludens or claudin-5. Moreover, the BBB function reveals increased permeability and leukocyte adhesion in cells expressing SSAO/VAP-1, as well as an enhancement of the vascular Aß deposition induced by mechanisms both dependent and independent of the enzymatic activity of SSAO/VAP-1. These results reveal an interesting role of vascular SSAO/VAP-1 in BBB dysfunction related to AD progression, opening a new window in the search of alternative therapeutic targets for fighting AD.