Saghir, A. | Search bioRxiv

bioRxiv2026

Fatty Acids Synergize with Bacteriophages Against Multidrug-Resistant Pathogens

In the face of rising antimicrobial resistance (AMR), phage therapy presents a promising alternative to antibiotics, but efficacy is limited by biofilms, narrow host range, and hosts rapidly developing phage resistance. Here we present a new concept for phage-based antimicrobials: using small-molecules to help phages infect and kill bacteria. Using checkerboard synergy assays, growth curves, and biofilm assays, we evaluate short- and medium-chain fatty acids (SCFAs/MCFAs) with diverse lytic phages against 18 strains of Stenotrophomonas maltophilia and extend our analysis to include more than 120 clinical isolates of Staphylococcus aureus, Enterococcus spp., and Pseudomonas aeruginosa. pH-neutral MCFAs (C6-C10) are bactericidal, do not diminish phage viability, and synergized with phages to delay or prevent phage-resistant bacterial rebound over 48 h. MCFAs lowered both attached and free bacterial biomass and were synergistic with envelope-active antibiotics (polymyxin B, ceftazidime, meropenem), supporting a mechanism involving bacterial envelope disruption. Phage-MCFA cooperativity was observed across both Gram-negative and Gram-positive bacteria: across 18 S. maltophilia strains and four lytic phages, phage-MCFA combinations were predominantly additive-to- synergistic; in 91 strains of Enterococcus spp., pH-neutral C8 produced widespread synergy with phage CAR, and enabled killing of strains recalcitrant to phage alone; in 18 strains of S. aureus, effects were stoichiometry-dependent, showing both synergy and antagonism depending on the ratio of phage-to- MCFA; in 14 strains of P. aeruginosa, interactions were largely additive. Together, this work elevates MCFAs as potential therapeutic accompaniments that stabilize and broaden phage efficacy, and highlights the general potential of small molecules as phage adjuvants.

microbiology↗

bioRxiv2022

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes.

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large populations wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

microbiology↗