Current antibiotics cannot eradicate uropathogenic Escherichia coli (UPEC) biofilms, leading to recurrent urinary tract infections. Here, we show that the insect antimicrobial peptide cecropin A (CecA) can destroy planktonic and sessile biofilm-forming UPEC cells, either alone or when combined with the antibiotic nalidixic acid (NAL), synergistically clearing infection in vivo without off-target cytotoxicity. The multi-target mechanism of action involves outer membrane permeabilization followed by biofilm disruption triggered by the inhibition of efflux pump activity and interactions with extracellular and intracellular nucleic acids. These diverse targets ensure that resistance to the CecA + NAL combination emerges slowly. The antimicrobial mechanisms of CecA, thus, extend beyond pore-forming activity to include an unanticipated biofilm-eradication process, offering an alternative approach to combat antibiotic-resistant UPEC infections.

Uropathogenic Escherichia coli (UPEC) strains cause symptomatic urinary tract infections in humans whereas commensal-like E. coli strains in the urinary bladder cause long-term asymptomatic bacteriuria (ABU). We previously reported that UPEC and ABU strains differentially regulate key DNA methylation and histone acetylation components in the surrogate insect host Galleria mellonella to epigenetically modulate innate immunity-related gene expression, which in turn controls bacterial growth. In this follow-up study, we infected G. mellonella larvae with UPEC strain CFT073 or ABU strain 83972 to identify differences in the expression of microRNAs (miRNAs), a class of non-coding RNAs that regulate gene expression at the post-transcriptional level. Our small RNA sequencing analysis showed that UPEC and ABU infections caused significant changes in the abundance of miRNAs in the larvae, and highlighted the differential expression of 147 conserved miRNAs and 95 novel miRNA candidates. We annotated the G. mellonella genome sequence to investigate the miRNA-regulated expression of genes encoding antimicrobial peptides, signaling proteins, and enzymatic regulators of DNA methylation and histone acetylation in infected larvae. Our results indicate that miRNAs play a role in the epigenetic reprograming of innate immunity in G. mellonella larvae to distinguish between pathogenic and commensal strains of E. coli. Urinary tract infections (UTIs) are a global public health problem, with 50% of all women experiencing a symptomatic UTI episode at least once in their lifetime. This results in 11 million medical visits and 100,000 hospital admissions in the United States every year 1,2. Uropathogenic Escherichia coli (UPEC) strains cause 70-90% of all UTIs in humans, and antibiotics are the front-line treatment option despite growing resistance among the target strains. UPEC strains infect the urinary bladder through the urethra (cystitis), and if they remain untreated, the infection can spread to the kidneys (pyelonephritis) leading to renal failure and sepsis. Unlike UPEC strains, commensal-like E. coli strains can colonize the urinary bladder in large numbers without symptoms. Such asymptomatic bacteriuria (ABU) strains have evolved from UPEC strains by losing the ability to express functional virulence factors 3-6. The ABU E. coli strain 83972 achieves long-term growth in the urinary bladder by adopting a commensal-like lifestyle. It blocks disease-associated signaling pathways and prevents symptomatic UTIs caused by more virulent UPEC strains 7-9. Innate immunity-related gene expression distinguishes between infections caused by ABU and UPEC strains in the urinary bladder. Bacterial molecular recognition patterns frequently expressed by bacterial pathogens activate different signaling pathways involved in innate immune response. Toll-like receptor (TLR) 4-mediated signaling distinguishes pathogenic from commensal strains and controls the downstream signaling pathways thus