Bacterial biofilms are surface-attached communities of slow- or non-replicating cells embedded within a protective matrix of biomolecules. Unlike free-floating planktonic bacteria, biofilms are innately tolerant to conventional antibiotics and are prevalent in recurring and chronic infections. Nitroxoline, a broad-spectrum biofilm-eradicating agent, was used to probe biofilm viability. Transcript profiling (RNA-seq) showed that 452 of 2594 genes (17.4%) in methicillin-resistant Staphylococcus aureus (MRSA) biofilms were differentially expressed after a 2 h treatment of nitroxoline. WoPPER analysis and time-course validation (RT-qPCR) revealed that gene clusters involved in iron acquisition (sbn, isd, MW2101, MW0695, fhu, and feo) were rapidly up-regulated following nitroxoline treatment, which is indicative of iron starvation in MRSA biofilms. In addition, genes related to oligopeptide transporters and riboflavin biosynthesis were found to be up-regulated, while genes related to carotenoid biosynthesis and nitrate assimilation were down-regulated. RT-qPCR experiments revealed that iron uptake transcripts were also up-regulated in established Staphylococcus epidermidis and Acinetobacter baumannii biofilms following nitroxoline treatment. Overall, we show RNA-seq to be an ideal platform to define cellular pathways critical for biofilm survival, in addition to demonstrating the need these bacterial communities have for iron.
Keywords: RNA-sequencing; bacterial biofilms; biofilm-eradicating agents; nitroxoline; transcriptomic analysis.