Biology subjects

Sanchez-Osuna, M.

Publications and source records attributed to Sanchez-Osuna, M..

8 recordsLinked to original sources

bioRxiv2026

Widespread occurrence of ampicillin-susceptible Enterococcus faecium and Enterococcus lactis clinical isolates with low MICs to cephalosporins from Spain and Portugal

Reduced cephalosporin resistance in Enterococcus faecium has traditionally been reported in laboratory mutants and, more recently, in a single clinical ampicillin-susceptible (AmpS) isolate. Herein, we investigated whether this phenotype is widespread by analysing 95 clinical enterococcal isolates (78 AmpS and 17 ampicillin resistant [AmpR]) collected from three hospitals in Spain and Portugal (2009-2025). Low ceftriaxone MICs ([≤]4 mg/L) were detected in 19/51 (37.3%) AmpS E. faecium and 7/27 (25.9%) E. lactis but in none of the AmpR isolates. Low ceftriaxone MICs were associated with older patient age in both species and with prior ampicillin therapy in E. faecium, but not with other clinical or epidemiological variables. Ceftaroline MICs were consistently low among AmpS isolates, while ceftriaxone and cefotaxime showed greater variability. Low-MIC isolates were distributed across multiple clonal lineages and hospitals and did not share a distinctive resistance or virulence gene profile. PBP5 phylogeny and variation at the psr-pbp5 region separated AmpS from AmpR E. faecium but did not explain variability in ceftriaxone MICs. Five AmpS isolates with reduced ceftriaxone MICs carried chromosomal deletions that included the psr-pbp5 region and genes with diverse cellular functions. Variation in other candidate resistance genes (pbpA, ponA, pbpF, croRS, stpA/stk and murAA) did not consistently explain the MIC differences. These results reveal unexpected heterogeneity in intrinsic cephalosporin resistance in clinical E. faecium and E. lactis and suggest that additional genetic or regulatory mechanisms underlie reduced susceptibility.

microbiology↗

bioRxiv2026

A Chemical-Genetic Interaction Matrix Reveals Drug Mechanism and Genetic Architecture

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

systems biology↗

bioRxiv2025

Single point mutations in global regulatory genes restore cephalosporin resistance in a low-MIC Enterococcus faecium natural isolate

Enterococcus faecium exhibits intrinsic resistance to cephalosporins (CPH), yet the genetic determinants of this phenotype remain incompletely understood. To date, E. faecium strains with low minimum inhibitory concentrations (MICs) to CPH have only been described following genetic manipulation. At Parc Tauli University Hospital, we identified a clinical isolate of ampicillin-susceptible E. faecium (Efm5) that exhibited unusually low-MICs to cefotaxime (1 mg/L), ceftriaxone (3 mg/L), and ceftaroline (0.19 mg/L). Upon single exposure to ceftriaxone (100 mg/L), Efm5 rapidly yielded variants with markedly increased MICs to ceftriaxone (>256 mg/L) and cefotaxime (>32 mg/L), while MICs to ampicillin and ceftaroline were unaffected. Whole-genome sequencing revealed that the high-MIC variants carried single nucleotide polymorphisms (SNPs) leading to non-synonymous mutations in croS, nusG or rpoB genes. Phenotypic assays confirmed that these mutations were associated with ceftriaxone resistance and immunoblots revealed increased expression of penicillin-binding protein 5 (PBP5) in all the high-MIC variants. Transcriptional profiling showed upregulation of the pbp5 operon, which includes ftsW, psr and pbp5, in the croS variants. This study provides the first evidence that E. faecium isolates with low-MICs to CPH can arise in clinical settings without laboratory manipulation, challenging the prevailing notion of absolute intrinsic CPH resistance in this species and offering a novel framework to explore previously unrecognized resistance pathways.

microbiology↗

bioRxiv2025

A Saccharomyces boulardii synthetic biotic platform for delivery of therapeutic nanobodies to ameliorate gastrointestinal inflammation

Protein-based pharmaceuticals, such as engineered antibodies, form a major drug class of steadily increasing market share. However, these biologic medicines are costly to manufacture, are subject to strict supply chain and storage constraints and often require invasive administration routes. Engineered microbes that secrete bioactive products directly within the microbiome milieu may mitigate these challenges. Here, we describe a cell microfactory platform based on the probiotic yeast Saccharomyces boulardii for the production of nanobody biologics in the gastrointestinal (GI) tract. High-level secretion of nanobodies by S. boulardii was achieved by optimizing promoters, secretion signals, and antibody formats. In mice, oral gavage of S. boulardii allowed efficient and transient colonization of the colonic compartment and in situ production of a therapeutic nanobody directed against tumor necrosis factor (TNF). In a mouse model of chemical-induced colitis, GI-delivery of anti-mTNF nanobody via live S. boulardii improved both survival and disease severity without causing overt perturbation of microbiome composition. These results position S. boulardii as a synthetic biotic platform for the in situ production and delivery of protein-based therapeutics to the GI tract.

synthetic biology↗

bioRxiv2025

Comparative mortality of dominant Staphylococcus aureus lineages in human bacteremia and animal infection models

Staphylococcus aureus is a major cause of severe infections including infective endocarditis, but lineage-specific virulence determinants remain unclear. We analyzed 77 S. aureus bacteremic isolates from major lineages using phenotypic assays, infection models, and transcriptomics. Our results revealed significant heterogeneity in S. aureus pathogenicity. ST398 isolates exhibited heightened virulence, characterized by increased hemolysin production, whereas CC30 strains showed reduced growth, biofilm formation, and infectivity. Notably, the ST398 agrC mutant Sau7 exhibited unique phenotypic behavior, with high biofilm production and decreased virulence in Galleria mellonella larvae model. Infection studies in the rabbit experimental endocarditis model showed increased vegetation size and bacterial load in Sau7-infected animals, highlighting the role of agr system in S. aureus colonization and biofilm formation. Transcriptomic analysis identified key pathways, including quorum sensing systems and hemolysins, driving virulence in ST398 strains. These findings provide insights into the lineage-specific virulence mechanisms and the multifaceted nature of S. aureus pathogenicity.

microbiology↗

bioRxiv2023

Genomic analysis of Staphylococcus aureus isolates from bacteremia reveals genetic features associated with the COVID-19 pandemic

Genomic analyses of bacterial isolates are necessary to monitor the prevalence of antibiotic resistance genes and virulence determinants. Herein, we provide a comprehensive genomic description of a collection of 339 Staphylococcus aureus strains isolated from patients with bacteremia between 2014 and 2022. Nosocomial acquisition accounted for 56.6% of episodes, with vascular catheters being the predominant source of infection (31.8%). Cases of fatality (27.4%), persistent bacteremia (19.5%) and diagnosis of septic emboli (24.2%) were documented. During the COVID-19 pandemic, we observed a 140% increase of the episodes of S. aureus bacteremia per year, with a concomitant increase of the cases from nosocomial origin. This prompted us to investigate the existence of genetic features associated with S. aureus isolates from the COVID-19 pandemic. While genes conferring resistance to {beta}-lactams (blaI-blaR-blaZ), macrolides (ermA, ermC, ermT, mphC, msrA) and aminoglycosides (ant(4)-Ia, ant(9)-Ia, aph(3)-IIIa, aph(2)-Ih) were prevalent in our collection, detection of the msrA and mphC genes increased significantly in pandemic S. aureus isolates. Similarly, we observed a higher prevalence of isolates carrying the genes encoding the Clumping Factors A and B, involved in fibrinogen binding. Of note, macrolides were extensively used as accessory therapy for COVID-19 and fibrinogen levels were usually elevated upon SARS-CoV-2 infection. Therefore, our results reveal a remarkable adaptation of the S. aureus isolates to the COVID-19 pandemic context and demonstrates the potential of whole-genome sequencing to conduct molecular epidemiology studies.

genomics↗

bioRxiv2020

Recurrent mobilization of ancestral and novel variants of the chromosomal di-hydrofolate reductase gene drives the emergence of clinical resistance to trimethoprim

2.Trimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydro-pteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen Acinetobacter baumannii, and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologs in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. This work hence confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants. 3. Impact statementAntibiotic resistance is a pressing and global phenomenon. It is well-established that resistance to conventional antibiotics emerged millions of years ago in either antibiotic-producing bacteria or their competitors. Resistance to synthetic chemotherapeutic agents cannot be explained by this paradigm, since these drugs are not naturally produced. Resistance is hence assumed to have evolved rapidly following the clinical introduction of these drugs. Recently we showed that resistance to one such drug, sulfonamide, evolved not recently, but millions of years ago, suggesting that the diversity of bacterial genomes may well contain genes conferring resistance to drugs yet to be developed. Here we analyze the origin of resistance to trimethoprim, another chemotherapeutic agent developed in the 1960s. Using phylogenetic methods, we identify new variants of the trimethoprim resistance genes that had not previously been reported, and we trace the chromosomal origins for a number of already known resistance variants. Our results show that resistance to trimethoprim is very diverse and has originated both from recent mutations and from preexisting ancient variants. These results stress the importance of gene mobilization mechanisms as the main drivers of the current antibiotic resistance phenomenon. 4. Data summary- The scripts used for data collection and analysis can be obtained at the GitHub ErillLab repository (https://github.com/ErillLab/). - The Bayesian phylogenetic tree can be visualitzed online on iTOL (https://itol.embl.de/tree/855674159451585133078) [1]. - The authors confirm all other supporting data has been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2018

Origin of the mobile di-hydro-pteroate synthase gene determining sulfonamide resistance in clinical isolates

Sulfonamides are synthetic chemotherapeutic agents that work as competitive inhibitors of the di-hydro-pteroate synthase (DHPS) enzyme, encoded by the folP gene. Resistance to sulfonamides is widespread in the clinical setting and predominantly mediated by plasmid- and integron-borne sul1-3 genes encoding mutant DHPS enzymes that do not bind sulfonamides. In spite of their clinical importance, the genetic origin of sul1-3 genes remains unknown. Here we analyze sul genes and their genetic neighborhoods to uncover sul signature elements that enable the elucidation of their genetic origin. We identify a protein sequence Sul motif associated with sul-encoded proteins, as well as consistent association of a phosphoglucosamine mutase gene (glmM) with the sul2 gene. We identify chromosomal folP genes bearing these genetic markers in two bacterial families: the Rhodobiaceae and the Leptospiraceae. Bayesian phylogenetic inference of FolP/Sul and GlmM protein sequences clearly establishes that sul1-2 and sul3 genes originated as a mobilization of folP genes present in, respectively, the Rhodobiaceae and the Leptospiraceae, and indicate that the Rhodobiaceae folP gene was transferred from the Leptospiraceae. Analysis of %GC content in folP/sul gene sequences supports the phylogenetic inference results and indicates that the emergence of the Sul motif in chromosomally-encoded FolP proteins is ancient and considerably predates the clinical introduction of sulfonamides. In vitro assays reveal that both the Rhodobiaceae and the Leptospiraceae, but not other related chromosomally-encoded FolP proteins confer resistance in a sulfonamide-sensitive Escherichia coli background, indicating that the Sul motif is associated with sulfonamide resistance. Given the absence of any known natural sulfonamides targeting DHPS, these results provide a novel perspective on the emergence of resistance to synthetic chemotherapeutic agents, whereby preexisting resistant variants in the vast bacterial pangenome may be rapidly selected for and mobilized upon the clinical introduction of novel chemotherapeuticals.

microbiology↗