Biology subjects

Rodriguez-Rojas, A.

Publications and source records attributed to Rodriguez-Rojas, A..

10 recordsLinked to original sources

bioRxiv2025

Speed Matters: Accelerated Pharmacokinetics Reduce Persisters in Escherichia coli

Persister cells are bacterial cells that form small subpopulations and are refractory to antimicrobials. Importantly, those cells can cause the relapse of an infection and also provide a starting point for antimicrobial resistance evolution. By now, several mechanisms of persister formation have been studied. For example, it has been shown that a short exposure to sublethal concentrations of antimicrobials can induce persister formation. Given this, we hypothesised that the pharmacokinetics, the temporal changes of drug concentrations under treatment, could impact persister formation. We predicted that the longer bacteria spend under sublethal drug concentrations, the more persisters would form. Using a set-up with small chemostats and the antimicrobial peptide Pexiganan, we indeed found that faster pharmacokinetics resulted in lower persister numbers compared to slower pharmacokinetics. This finding provides a proof of principle that pharmacokinetics, which can be influenced by treatment procedures, has consequences for persister formation. Our results suggest that faster pharmacokinetics could be useful to minimize persister numbers and hence to lower the risks of relapse or resistance evolution.

evolutionary biology↗

bioRxiv2024

Cross-linked agarose-gelatine beads as a substrate for investigating biofilms of bacterial pathogens

Treating chronic bacterial infections is challenging due to the formation of biofilms, making bacteria less susceptible to antimicrobials. In vitro models have limitations in replicating biofilm physiology. To address this problem, we have created a hydrogel substrate that combines crosslinked agarose and gelatine presented as beads, providing stability and resistance to autoclaving. Bacterial pathogens rapidly colonise these biogel beads when submerged in liquid culture. The substrate was tested with Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showing more robust biofilm growth than its glass bead counterpart. Additionally, this led to increased virulence factor production and served as a reservoir for biofilm quorum sensing molecules. These features closely resemble clinical situations, suggesting a more accurate representation of biofilm-associated infections than current approaches. This new substrate offers a practical and convenient model for studying biofilms of bacterial pathogens, providing an efficient solution to the research community and holding promise for future breakthroughs.

microbiology↗

bioRxiv2023

Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a low susceptible strain of Aeromonas hydrophila, namely INISA09, to from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and determined the primary mechanisms related to its resistance using genomic and proteomic tools. The genome of the strain is 4.6-Mb with 4,273 genes, and we found a propound genome rearrangement and thousands of missense mutations when compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, quantitative proteomic analysis revealed the significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings suggest that the response of A. hydrophila INISA09 to BAC primarily occurs at the membrane level, which is BACs primary target. This study sheds light on the mechanisms of antimicrobial susceptibility in tropical aquatic environments against a widely-used disinfectant, using an environmental strain that could serve as a new study model.

microbiology↗

bioRxiv2022

Antibiotic-induced recombination in Gram-negative bacteria requires the formation of double-strand breaks

Recombination is an essential process in bacterial drug resistance evolution. Fluoroquinolones, an important class of antibiotics, are known to stimulate recombination in Escherichia coli. When bacteria are exposed to antibiotics other than fluoroquinolones, including strong up-regulators of recombination pathways, there is no detectable change in the recombination level. Here we explore why fluoroquinolones, but not other antimicrobials, increase recombination rates. Fluoroquinolones, in contrast to other antibiotics, generate DNA double-strand breaks (DSBs). We tested whether other drugs that also cause double-strand breaks, such as mitomycin C and bleomycin, also affect bacterial recombination rates with consistent increases in recombination. A positive correlation between the number of DSBs and the recombination frequency was found. The manipulation of the level of DSBs directly impacted the recombination frequency. Our results highlight that only antibiotics that induce DNA double-strand breaks are more probable to increase genetic diversity via recombination. The stimulation of recombination by DSB-causing antimicrobials is an additional factor leading to the risk of antibiotic resistance evolution.

microbiology↗

bioRxiv2020

Amino acid oxidation constrained protein sequences during evolution: an analysis of a model bacterium.

Within the cell, there are compartments and local conditions that may constrain amino acid sequence. Life emerged in an anoxic world but releasing a photosynthesis by-product, the molecular oxygen, forced adaptive changes to counteract its toxicity. Several mechanisms evolved to balance the intracellular redox state and maintain a reductive environment more compatible with many essential biological functions. Here, we statistically interrogate the aminoacidic composition of E. coli proteins and investigate how the proneness or susceptibility to oxidation of amino acid biased their sequences. By sorting the proteins in five different compartments: cytoplasm, internal membrane, periplasm, outer membrane and extracellular; we found that various oxidative lesions constrain protein composition, that there is a dependency of the cellular compartments and there is an impact on the evenness distribution or frequency. This analysis is necessary from an evolutionary point of view to reflect how the oxidative atmosphere could restrict protein composition and probably imposed a trend in the codon bias.

evolutionary biology↗

bioRxiv2020

Antimicrobial peptide induced-stress renders Staphylococcus aureus susceptible to toxic nucleoside analogues

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty to reach sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defence peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied if sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that S. aureus is highly susceptible to antimetabolite nucleoside analogues when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.

microbiology↗

bioRxiv2019

Seawater salt-trapped Pseudomonas aeruginosa survives for years and gets primed for salinity tolerance

BackgroundIn nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood.\n\nResultsIn this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after fourteen years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that seventy-eight genes had differential expression between the parental strain and derivative clones. Sixty-one transcripts were up-regulated, while seventeen were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions.\n\nConclusionsThe individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.

microbiology↗

bioRxiv2017

Genomic signature of experimental adaptation of Staphylococcus aureus to a natural combination of insect antimicrobial peptides

Antimicrobial peptides are highly conserved immune effectors across the tree of life and are employed as combinations. In the beetle Tenebrio molitor, a defensin and a coleoptericin are highly expressed in vivo after inoculation with S. aureus. The defensin displays strong in vitro activity but no survival benefit in vivo. The coleoptericin provides a survival benefit in vivo, but no activity in vitro. To investigate this paradox we experimentally evolved S. aureus to increased resistance against the defensin and a combination of the defensin and coleoptericin. Genome re-sequencing showed that resistance was associated with mutations in either the ytr or nsa operons, in both AMP treatments. Strains with these mutations show longer lag phases, slower Vmax and nsa mutants reach lower final population sizes. Mutations in rpoB were showed a further increase in the lag phase in nsa mutants but not in ytr mutants. In contrast, final MICs do not segregate by mutation. All resistant lines display AMP but not antibiotic cross-resistance. Costly resistance against AMPs readily evolves for an individual AMP as well as a naturally occurring combination in vitro and provides broad protection against AMPs. Such non-specific resistance could result in strong selection on host immune systems that rely on cocktails of AMPs.

evolutionary biology↗

bioRxiv2017

Clay-induced DNA double-strand breaks underlay genetic diversity, antibiotic resistance and molecular basis of asbestosis

Some natural clays and synthetic nanofibres present in the environment have a severe impact on human health. After several decades of research, the molecular mechanism of how asbestos induce cancers is not well understood. Different fibres, including asbestos, can penetrate the membrane and introduce DNA in both, bacterial and eukaryotic cells. Incubating Escherichia coli with sepiolite, a clayey material, and asbestos under friction forces, both fibres cause double-strand breaks in bacteria. Since antibiotics and clays are used together in animal husbandry, the mutagenic effect of these fibres might constitute a pathway to antibiotic resistance due to the friction provided by peristalsis of the gut from farm animals in addition to the previously proposed horizontal gene transfer. Moreover, we raise the possibility that the same mechanism could generate bacteria diversity in natural scenarios with a role in the evolution of species. Finally, we provide a new model on how asbestos may promote mutagenesis and cancer based on the observed mechanical genotoxicity.

evolutionary biology↗

bioRxiv2017

Demographic variability and heterogeneity among individuals within and among clonal bacteria strains

Identifying what drives individual heterogeneity has been of long interest to ecologists, evolutionary biologists and biodemographers, because only such identification provides deeper understanding of ecological and evolutionary population dynamics. In natural populations one is challenged to accurately decompose the drivers of heterogeneity among individuals as genetically fixed or selectively neutral. Rather than working on wild populations we present here data from a simple bacterial system in the lab, Escherichia coli. Our system, based on cutting-edge microfluidic techniques, provides high control over the genotype and the environment. It therefore allows to unambiguously decompose and quantify fixed genetic variability and dynamic stochastic variability among individuals. We show that within clonal individual variability (dynamic heterogeneity) in lifespan and lifetime reproduction is dominating at about 82-88%, over the 12-18% genetically (adaptive fixed) driven differences. The genetic differences among the clonal strains still lead to substantial variability in population growth rates (fitness), but, as well understood based on foundational work in population genetics, the within strain neutral variability slows adaptive change, by enhancing genetic drift, and lowering overall population growth. We also revealed a surprising diversity in senescence patterns among the clonal strains, which indicates diverse underlying cell-intrinsic processes that shape these demographic patterns. Such diversity is surprising since all cells belong to the same bacteria species, E. coli, and still exhibit patterns such as classical senescence, non-senescence, or negative senescence. We end by discussing whether similar levels of non-genetic variability might be detected in other systems and close by stating the open questions how such heterogeneity is maintained, how it has evolved, and whether it is adaptive.\n\nData depositionThe processed image analysis data, R code, as well as the Leslie matrices will be archived at Dryad.org.

evolutionary biology↗