Biology subjects

Laborda, P.

Publications and source records attributed to Laborda, P..

9 recordsLinked to original sources

bioRxiv2026

Infection-dependent antibiotic response phenotypes emerge during chronic airway adaptation of Pseudomonas aeruginosa

Objectives Chronic Pseudomonas aeruginosa airway infections can persist for years despite intensive antibiotic therapy guided by standard antimicrobial susceptibility testing. While this technique captures the emergence of resistance, it does not reproduce many features of the infection environment, where adaptation acquired during chronic infection may alter bacterial responses to antibiotics in ways that are not fully reflected by conventional susceptibility testing. Here we investigated antibiotic responses of longitudinal clinical isolates under standard laboratory conditions and in an airway epithelial infection model. Methods Six longitudinal pairs of P. aeruginosa isolates from people with cystic fibrosis were analysed. Changes in bacterial growth following exposure to tobramycin, ceftazidime and ciprofloxacin were quantified under standard laboratory conditions and compared with changes in bacterial burden after treatment measured in differentiated airway epithelial cultures maintained under air-liquid interface (ALI) conditions that reproduce key features of the airway infection environment. Representative phenotypes were visualized by confocal microscopy and interpreted in the context of available whole-genome sequencing data. Results Major resistance phenotypes identified by conventional susceptibility testing were largely reproduced in the ALI model. However, concordance between phenotypes observed under standard laboratory and ALI conditions differed between antibiotics. Several isolates classified as susceptible according to conventional susceptibility testing displayed substantial survival in the ALI model, and these discordant phenotypes were observed predominantly among those isolates recovered after prolonged airway persistence. Genomic analyses identified mutations associated with both classical resistance mechanisms and chronic adaptation. Conclusions Phenotypes observed under conventional laboratory conditions do not uniformly predict those observed in the ALI airway conditions. Discordant phenotypes were most apparent among chronically adapted isolates and varied between antibiotics, suggesting that adaptation acquired during chronic infection can alter antibiotic responses in ways that are not fully reflected by conventional susceptibility testing.

microbiology↗

bioRxiv2026

Exploiting outer-membrane protein promiscuity to induce transient collateral sensitivity via efflux pump competition

In the fight against antimicrobial resistance, the identification of robust collateral sensitivity (CS) patterns could form the basis of successful sequential and combinatorial antimicrobial therapies. While most of the focus has been devoted to study stable CS due to mutations, CS can be transiently induced in Pseudomonas aeruginosa using dequalinium chloride (DC), which increases the expression of the efflux operon mexCD-oprJ. This leads to transient CS to the aminoglycoside tobramycin, whose molecular mechanism remains unclear. Using a combination of experimental results, mechanistic mathematical models and structural simulations, we show that the inactivation of NfxB by DC not only increases mexCD expression but also reduces the effective amount of the MexXY-OprM aminoglycosides efflux pump. Our data suggest that, under conditions of high MexCD production, this efflux pump can outcompete MexXY for the outer-membrane protein OprM, thereby inducing CS to tobramycin. Bayesian fits accurately reproduce measured mexCD expression and minimal inhibitory concentration (MIC) shifts across DC doses. Model predictions were validated in P. aeruginosa PA14 mutants lacking functional outer-membrane channels. Loss of OprJ preserves DC-induced CS to tobramycin, whereas loss of OprM abolishes it. These results identify competition for OprM as the proximal cause of DC-induced, transient CS and provide a generalizable framework for eliciting CS by perturbing shared components of multidrug efflux systems. This strategy suggests immediate avenues to enhance aminoglycoside efficacy while minimizing selection for stable resistance.

microbiology↗

bioRxiv2026

Host-microbe-immune interactions in an air-liquid interface airway model

BackgroundAir-liquid interface (ALI) cell culture systems have improved the study of host-microbe interactions in respiratory infections. However, most ALI models lack immune components, limiting their ability to capture epithelial-immune crosstalk. To address this, we developed a dual-cell ALI model incorporating human peripheral blood monocyte-derived macrophages beneath differentiated airway epithelial cells. MethodologyMacrophages were seeded on the basolateral side of transwell inserts using fibronectin coating. Model characterization included transepithelial electrical resistance (TEER) to assess epithelial barrier integrity, IL-8 secretion as a marker of epithelial inflammatory signaling, and confocal microscopy to evaluate cellular architecture before and after infection. Mono-and dual-cell cultures were infected with the laboratory strain Pseudomonas aeruginosa PAO1. ResultsMacrophages adhered stably to the basolateral surface without compromising epithelial barrier integrity. Following infection, IL-8 secretion was elevated in epithelial monocultures compared to dual-cell cultures, suggesting early immune modulation in the presence of macrophages. While overall bacterial burden was comparable, confocal imaging revealed clustered bacterial growth in monocultures and a more dispersed spatial distribution in dual-cell cultures. ConclusionsThis dual-cell ALI model enables investigation of early epithelial-immune interactions, inflammatory modulation, and bacterial colonization dynamics during airway infection. The system provides a versatile and human-relevant platform for studying respiratory host-pathogen interactions.

cell biology↗

bioRxiv2025

Immune protection and disease tolerance in Pseudomonas aeruginosa infections by pyruvate cross-feeding

Chronic infections by Pseudomonas aeruginosa in people with cystic fibrosis are characterized by persistent inflammation and oxidative stress, yet the mechanisms enabling bacterial persistence are not fully understood. Here, we identify a novel persistence mechanism mediated by pyruvate secretion resulting from mutations in the pyruvate dehydrogenase complex in clinical isolates of P. aeruginosa, with analogous mutations also identified in Staphylococcus aureus, Haemophilus influenzae and Stenotrophomonas maltophilia. These mutations lead to elevated extracellular pyruvate which helps the establishment a more tolerogenic infection microenvironment and favor bacterial persistence. Pyruvate exerts multiple roles: scavenges reactive oxygen species such as H2O2, suppresses host immune activation both in airway epithelial cells and macrophages, and increases bacterial survival during phagocytosis. This metabolic crosstalk promotes bacterial persistence while limiting host tissue damage, aligning with the concept of disease tolerance. Our findings reveal pyruvate as a bacterial immunometabolite that mimics host antioxidant defenses, reshaping the infection niche to favor long-term colonization. This work highlights the broader role of secreted metabolites in host-pathogen interactions and suggests new strategies targeting metabolic pathways to manage chronic infections.

microbiology↗

bioRxiv2025

Carbapenem-resistance oprD mutations reshape Pseudomonas aeruginosa host-pathogen interactions during infection

Antibiotic resistance is a major global health threat. While its role in reducing drug susceptibility is well established, the broader consequences of resistance mutations on bacterial physiology and behavior during infection remain poorly understood. Carbapenem-resistant Pseudomonas aeruginosa is considered among the highest- priority bacterial threats, with resistance commonly driven by loss-of-function mutations in the carbapenem uptake porin OprD. Here we show that such mutations can arise in clinical isolates even without prior carbapenem treatment, indicating that their effects during infection extend beyond antibiotic resistance. Consistent with this, we found that oprD mutants exhibit enhanced early attachment to and translocation across airway epithelial barriers in infection models, an effect maintained across strains with distinct clinical genomic backgrounds and infection dynamics. Our findings indicate that loss of OprD alters the bacterial outer membrane charge and reduces mucus entrapment, thereby facilitating epithelial barrier colonization. Overall, these results illustrate how antibiotic resistance mutations can directly shape infection dynamics, extending their impact well beyond antimicrobial susceptibility.

microbiology↗

bioRxiv2025

Roles of spoVF operon subunits A and B in regulating cell morphology and biofilm formation in the biocontrol agent Bacillus subtilis

Enhanced expression of spoVF operon (sporulation initiation factor; dipicolinic acid (DPA) synthase) in Bacillus subtilis vegetative cells has been reported to increase host attachment and biocontrol ability. However, how the spoVF operon regulates these essential biocontrol factors remains unknown. In this study, it was found that the knockout of the spoVFA or spoVFB subunits, individually ({Delta}A and{Delta} B), or together ({Delta}A{Delta}B), decreased DPA biosynthesis, host attachment, nutrient competition ability, and biocontrol efficacy in B. subtilis. Moreover, the cell shape of the knockout strains changed from short rod to spherical. qRT-PCR analysis indicated that the spoVF operon regulates TCA cycle expression and biofilm formation by balancing the amounts of various transcriptional factors, such as spo0A, SinR, and AbrB, and the cofactor NADPH. The regulatory mechanism of the spoVF operon in B. subtilis biofilm-related genes was proposed for the first time. IMPORTANCEBacillus subtilis SpoVF operon contains the spoVFA or spoVFB subunits, which are responsible for the dipicolinic acid synthase activity. Dipicolinic acid is an important component of B. subtilis spores. In this study, the roles of spoVFA or spoVFB subunits in the biocontrol ability B. subtilis were examined, indicating that the spoVFA subunit plays a key role in B. subtilis biofilm formation, cell morphology, growth, and nutrient competition ability. Additionally, the regulatory network connecting the spoVF operon with biofilm formation-related genes of B. subtilis was proposed. The results obtained in this study help to understand key genes involved in the biocontrol ability of B. subtilis.

microbiology↗

bioRxiv2024

Macrolide resistance through uL4 and uL22 ribosomal mutations in Pseudomonas aeruginosa

Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media, Pseudomonas aeruginosa is considered intrinsically resistant and, therefore, antibiotic susceptibility testing against macrolides is not performed. Nevertheless, due to macrolides immunomodulatory effect and suppression of virulence factors, they are used for the treatment of persistent P. aeruginosa infections. Here, we demonstrate that macrolides are, instead, effective antibiotics against P. aeruginosa airway infections in an air-liquid interface (ALI) infection model system resembling the human airways. Importantly, macrolide treatment in both people with cystic fibrosis and primary ciliary dyskinesia patients leads to the accumulation of uL4 and uL22 ribosomal protein mutations in P. aeruginosa which causes antibiotic resistance. Consequently, higher concentrations of antibiotics are needed to modulate the macrolide-dependent suppression of virulence. Surprisingly, even in the absence of antibiotics, these mutations also lead to a collateral reduction in growth rate, virulence and pathogenicity in airway ALI infections which are pivotal for the establishment of a persistent infection. Altogether, these results lend further support to the consideration of macrolides as de facto antibiotics against P. aeruginosa and the need for resistance monitoring upon prolonged macrolide treatment.

microbiology↗

bioRxiv2023

Efflux pump mutations in Pseudomonas aeruginosa cause low-level clinical resistance and high-level tolerance to antibiotics in patients

Pseudomonas aeruginosa is one of the microorganisms with high-risk regarding antimicrobial resistance, since it has an overwhelming capacity to acquire antibiotic resistance, mainly by mutations during persistent lung infections. Mutations in mexZ, encoding the local negative regulator of genes encoding the MexXY efflux pump, are very frequently acquired at early stages of P. aeruginosa infections, while they are rarely selected for in vitro. Although traditionally related to resistance to the first-line drug tobramycin, caused by the overproduction of the aminoglycosides MexXY efflux pump, mutations in mexZ are actually associated with low levels of aminoglycosides resistance when determined in the clinical microbiology laboratory. This very moderate but frequent phenotype suggests that these mutations may shape the infection process, beyond causing conventional resistance. Here we investigated the colonization strategy of a mexZ mutant, compared to a wild-type strain, in a human airway infection model. We observed that the mexZ mutant tends to accumulate inside the epithelial cell layer. This behaviour allows bacteria to colonise the epithelium while being more protected against diverse antibiotics. The altered colonization phenotype was caused by the overexpression of lecA, a Quorum Sensing regulated gene encoding a lectin involved in P. aeruginosa tissue invasiveness. lecA upregulation was underlied by the competition for the shared porin, OprM, between the overproduced MexXY and the MexAB efflux pump, responsible for extruding Quorum Sensing molecules. These findings suggest that standardised antimicrobial susceptibility determined in the clinic may be misleading because antibiotic resistance often depends on the infection environment.

microbiology↗

bioRxiv2019

Novel inducers of the expression of multidrug efflux pumps that trigger Pseudomonas aeruginosa transient antibiotic resistance

The study of the acquisition of antibiotic resistance (AR) has mainly focused in inherited processes, namely mutations and acquisition of AR genes. However, inducible, non-inheritable AR has received less attention and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about non-antibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tightly regulation of their expression. Their contribution to acquired AR relies in their overexpression. Herein we analyzed potential inducers of the expression of the chromosomally-encoded Pseudomonas aeruginosa clinically-relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able of modifying the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog Phenotype Microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of mexCD-oprJ. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine or atropine are present. This work emphasizes the need of studying compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.

microbiology↗