María Escobar-Salom#, 1, 2, 3 , Isabel María Barceló#, 1, 2, 3, Estrella Rojo-Molinero1, 2, 3, Elena Jordana-Lluch1, 2, 3, Gabriel Cabot1, 2, 3 , Antonio Oliver1, 2, 3, Carlos Juan1, 2, 3
1 Research Unit, University Hospital Son Espases-Health Research Institute of the Balearic Islands (IdISBa), Palma, Spain.
2 Microbiology Department, University Hospital Son Espases, Palma, Spain.
3 Centro de Investigación Biomédica en Red en Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain.
# Contributed equally
Abstract:
The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC.
Importance:
In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).
Keywords:
Acinetobacter baumannii; ESKAPE Gram-negatives; Enterobacter cloacae complex; Human Neutrophil Peptide-1 (HNP-1); Klebsiella pneumoniae; Pseudomonas aeruginosa; human Beta Defensin 3 (hBD-3); peptidoglycan recycling.
Microbiol Spectrum. 2024 Mar 5;e0035824. doi: 10.1128/spectrum.00358-24. Online ahead of print.
Antibiotic Resistance and Pathogenicity of Bacterial Infections Group 
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