Komi, M. | bioRxiv Science

bioRxiv2026

Distinct plasmid- and host-encoded mechanisms drive small plasmid copy number-mediated heteroresistance in Escherichia coli

Antibiotic heteroresistance, the presence of a rare resistant subpopulation within an otherwise susceptible bacterial population, poses a significant clinical challenge. Understanding its genetic mechanisms is critical for early detection and treatment efficacy. Here, we investigate the contribution of small plasmids to heteroresistance using a clinical bloodstream Escherichia coli isolate carrying a 12 kb ColE1-type plasmid (p12). We show that this plasmid drives transient {beta}-lactam heteroresistance through massive increases in plasmid copy number. Two distinct genetic mechanisms drive this amplification: mutations in the plasmid RNAI/RNAII that deregulate replication control, and a chromosomal recD mutation that induces multimerization and a shift toward rolling-circle replication. Notably, this recD-mediated amplification is restricted to small ColE1 and F- plasmids. This study highlights the crucial role of small plasmids in resistance evolution, demonstrating that they can cause this phenotype via alternative genetic pathways, without the involvement of traditional large resistance plasmids.

microbiology↗

bioRxiv2025

The pcnB gene sustains Shigella flexneri virulence

The enteropathogen Shigella flexneri employs a Type Three Secretion System (T3SS) to colonize intestinal epithelial cells. Genes encoding the T3SS are located on a large IncFII virulence plasmid, pINV. T3SS expression comes at the expense of slowed Shigella growth and is therefore strictly controlled by both transcriptional and post-transcriptional mechanisms. Following up on a recent genome-wide screen, we here show that the chromosomal gene pcnB, encoding the poly-A polymerase I (PAP-I), slows Shigella growth at 37{degrees}C, while at the same time promotes early colonization of a human epithelial enteroid model. Proteomic profiling revealed that pcnB drives global increase of the Shigella T3SS virulence program. Accordingly, pcnB sustains pINV replication to a level optimal for Shigella virulence. This is achieved through increased degradation of the antisense RNA CopA, involved in plasmid replication control. The pcnB effect on pINV replication was found to also ensure longer-term intraepithelial expansion of Shigella following human intestinal epithelium invasion. Our findings exemplify how an optimal pINV level is necessary for the execution of Shigellas infection cycle. AUTHOR SUMMARYBacterial infections represent a major global threat. Understanding the genetic determinants promoting infections is crucial to overcome this threat. Shigella is an intracellular bacterial pathogen that invades and disseminates in the intestinal epithelium, causing bacillary dysentery in humans. Shigellas ability to cause disease relies on the delivery of effector proteins into the host cells through an injection machinery, with most of the genes involved in this process located on a large virulence plasmid. Here we show that the chromosomal gene pcnB sustains an optimal virulence plasmid level. This is crucial for Shigella to maximize virulence protein expression and thereby efficiently invade, replicate and spread within the intestinal epithelium.

microbiology↗