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bioRxiv2026

Comparative genomics of clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients in Mexico

Pseudomonas aeruginosa (P. aeruginosa) is the primary pathogen responsible for morbidity and mortality in patients with cystic fibrosis (CF). Its genomic plasticity and constant selective pressure from antimicrobial treatments have favored the emergence of multidrug-resistant clones. This study conducted a comparative genomic analysis of 41 P. aeruginosa isolated from pediatric patients with CF in Mexico from 2015 to 2024, with the aim of characterizing their evolutionary dynamics, resistome, and virulome. Whole-genome sequencing (MGI, Illumina, and PacBio platforms) was used, with de novo assemblies performed using Unicycler v0.4.8 on the BV-BRC platform. The databases used for the resistome were CARD and NDARO, and for the virulome, VFDB. Phylogenetic reconstruction was based on core-genome alignments generated with Roary v3.13.0, with maximum likelihood reconstruction performed in IQ-TREE v2.1.2. The statistical significance of the segregation of resistance and virulence patterns was evaluated using PERMANOVA analysis. The results revealed a significant clonal prevalence of sequence types (ST) 307 and ST 167. Phylogenomic analysis grouped the isolates into three main clades; Clade 1 stood out for having the highest resistance gene load (mean of 75 genes/genome), establishing itself as the main reservoir of multidrug-resistant profiles. Genotype-phenotype concordance reached 65.5% overall, with high accuracy for aminoglycosides (87.8%) and fluoroquinolones (82.9%). Furthermore, virulome analysis identified 67 distinct patterns that were significantly segregated among the clades (PERMANOVA: R2=0.31, p=0.001). These findings demonstrate that the evolution of P. aeruginosa lineages in the pediatric clinical setting involves parallel and coordinated adaptations in both their resistance potential and their virulence arsenal. This study underscores the need to adopt a multidisciplinary approach to the clinical management of chronic P. aeruginosa infections in pediatric patients. The persistence of extensively drug-resistant (XDR) strains calls for the integration of genomic surveillance and functional diagnostics, as well as the search for therapeutic alternatives for the clinical management of patients with cystic fibrosis.

microbiology↗

bioRxiv2026

Kaposi's sarcoma-associated herpesvirus forms and maintains R-loops at origins of lytic replication

GC-rich sequences are abundant in human herpesviruses genomes. GC-rich regions can form three-stranded RNA:DNA hybrid structures called R-loops. Though these hybrid structures serve important biological roles at telomeres or during cellular DNA synthesis, unscheduled or prolonged R-loop formation causes DNA damage and genome instability. For this reason, several mechanisms exist to resolve R-loops including endoribonucleases RNaseH1 (constitutively expressed) and RNaseH2A (cell cycle-regulated) which degrade the RNA portion of the R-loop. The Kaposi's sarcoma-associated herpesvirus (KSHV) origins of lytic replication (OriLyts) contain multiple cis-acting elements that are required for viral DNA replication including the production of GC-rich and repetitive transcripts, T1.4 (OriLyt-L) and kaposin (OriLyt-R). We previously showed that R-loops form at both OriLyts and that deleting kaposin repeats or decreasing their GC-rich content prevented R-loop formation at OriLyt-R, reduced genome amplification after primary infection and caused defects in latency establishment. To define the contribution that R-loops play in KSHV replication, we overexpressed RNaseH1, reasoning that excess RNaseH1 would resolve both OriLyt R-loops. However, RNaseH1 protein levels decreased following KSHV reactivation in both iSLK and BCBL-1 cell lines. Using co-transfection, we discovered that the KSHV viral replication and transcription activator protein, RTA, mediated RNaseH1 protein decreases in a E3 ligase domain-dependent manner without impacting levels of its cognate RNA transcript. We attempted to construct an RTA-resistant yet functional version of RNaseH1 by site-directed mutagenesis of lysine residues individually or in combination, yet these constructs remain susceptible to RTA-mediated protein decreases. An amino terminally tagged RNaseH1 displayed reduced susceptibility to RTA, suggesting that RTA may target the N-terminus of RNaseH1 for ubiquitination. However, overexpression of the cell-cycle regulated endonuclease, RNaseH2, exhibited RTA resistance, suggesting RNaseH2 may be a tool that will effectively resolve R-loops during KSHV infection. KSHV is not the only herpesvirus to encode a protein that reduces RNaseH1 levels, as co-expression of RTA homologs from the related gamma-herpesviruses EBV and MHV-68 likewise decreased steady-state levels of RNaseH1 protein. We propose that RTA-mediated RNaseH1 degradation is conserved strategy to ensure R-loop persistence during gamma-herpesvirus infection, underscoring the importance of these structures.

microbiology↗