Antibiotic resistance is a global public health issue of growing proportions. All antibiotics are susceptible to resistance. The evidence is now clear that the environment is the single largest source and reservoir of resistance. Soil, aquatic, atmospheric, animal-associated, and built ecosystems are home to microbes that harbor antibiotic resistance elements and the means to mobilize them. The diversity and abundance of resistance in the environment is consistent with the ancient origins of antibiotics and a variety of studies support a long natural history of associated resistance. The implications are clear: Understanding the evolution of resistance in the environment, its diversity, and mechanisms is essential to the management of our existing and future antibiotic resources.

Rifamycin monooxygenases (Rox) are present in a variety of environmental bacteria and are associated with decomposition of the clinically utilized antibiotic rifampin. Here we report the structure and function of a drug-inducible rox gene from Streptomyces venezuelae, which encodes a class A flavoprotein monooxygenase that inactivates a broad range of rifamycin antibiotics. Our findings describe a mechanism of rifamycin inactivation initiated by monooxygenation of the 2-position of the naphthyl group, which subsequently results in ring opening and linearization of the antibiotic. The result is an antibiotic that no longer adopts the basket-like structure essential for binding to the RNA exit tunnel of the target RpoB, thereby providing the molecular logic of resistance. This unique mechanism of enzymatic inactivation underpins the broad spectrum of rifamycin resistance mediated by Rox enzymes and presents a new antibiotic resistance mechanism not yet seen in microbial antibiotic detoxification.

The transition of the opportunistic pathogen Pseudomonas aeruginosa from free-living bacteria into surface-associated biofilm communities represents a viable target for the prevention and treatment of chronic infectious disease. We have established a proteomics platform that identified 2443 and 1142 high-confidence proteins in P. aeruginosa whole cells and outer-membrane vesicles (OMVs), respectively, at three time points during biofilm development (ProteomeXchange identifier PXD002605). The analysis of cellular systems, specifically the phenazine biosynthetic pathway, demonstrates that whole-cell protein abundance correlates to end product (i.e., pyocyanin) concentrations in biofilm but not in planktonic cultures. Furthermore, increased cellular protein abundance in this pathway results in quantifiable pyocyanin in early biofilm OMVs and OMVs from both growth modes isolated at later time points. Overall, our data indicate that the OMVs being released from the surface of the biofilm whole cells have unique proteomes in comparison to their planktonic counterparts. The relative abundance of OMV proteins from various subcellular sources showed considerable differences between the two growth modes over time, supporting the existence and preferential activation of multiple OMV biogenesis mechanisms under different conditions. The consistent detection of cytoplasmic proteins in all of the OMV subproteomes challenges the notion that OMVs are composed of outer membrane and periplasmic proteins alone. Direct comparisons of outer-membrane protein abundance levels between OMVs and whole cells shows ratios that vary greatly from 1:1 and supports previous studies that advocate the specific inclusion, or "packaging", of proteins into OMVs. The quantitative analysis of packaged protein groups suggests biogenesis mechanisms that involve untethered, rather than absent, peptidoglycan-binding proteins. Collectively, individual protein and biological system analyses of biofilm OMVs show that drug-binding cytoplasmic proteins and porins are potentially shuttled from the whole cell into the OMVs and may contribute to the antibiotic resistance of P. aeruginosa whole cells within biofilms.

Increasing antibiotic resistance among pathogenic bacterial species is a serious public health problem and has prompted research examining the antibacterial effects of alternative compounds and novel treatment strategies. Compounding this problem is the ability of many pathogenic bacteria to form biofilms during chronic infections. Importantly, these communities are often recalcitrant to antibiotic treatments that show effectiveness against acute infection. The antimicrobial properties of silver have been known for decades, but recently silver and silver-containing compounds have seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the ability of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the aminoglycoside antibiotic tobramycin, to inhibit established biofilms. Our results demonstrate that smaller 10-nm and 20-nm AgNPs were more effective at synergistically potentiating the activity of tobramycin. Visualization of biofilms treated with combinations of 10-nm AgNPs and tobramycin reveals that the synergistic bactericidal effect may be caused by disrupting cellular membranes. Minimum biofilm eradication concentration (MBEC) assays using clinical isolates shows that small AgNPs are more effective than larger AgNPs at inhibiting biofilms, but that the synergy effect is likely a strain-dependent phenomenon. These data suggest that small AgNPs synergistically potentiate the activity of tobramycin against and may reveal a potential role for AgNP/antibiotic combinations in treating patients with chronic infections in a strain-specific manner.

Microbial biofilms are particularly resistant to antimicrobial therapies. These surface-attached communities are protected against host defenses and pharmacotherapy by a self-produced matrix that surrounds and fortifies them. Recent proteomic evidence also suggests that some bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, undergo modifications within a biofilm that make them uniquely resistant compared to their planktonic (free-living) counterparts. This study examines 50 proteins in the resistance subproteome of both surface-associated and free-living P. aeruginosa PAO1 over three time points. Proteins were grouped into categories based on their roles in antimicrobial: (i) binding, (ii) efflux, (iii) resistance, and (iv) susceptibility. In addition, the extracellular outer membrane vesicle-associated proteome is examined and compared between the two growth modes. We show that in whole cells between 12–24% of the proteins are present at significantly different abundance levels over time, with some proteins being unique to a specific growth mode; however, the total abundance levels in the four categories remain consistent. In contrast, marked differences are seen in the protein content of the outer membrane vesicles, which contain a greater number of drug-binding proteins in vesicles purified from late-stage biofilms. These results show how the method of analysis can impact the interpretation of proteomic data (i.e., individual proteins vs. systems), and highlight the advantage of using protein-based methods to identify potential antimicrobial resistance mechanisms in extracellular sample components. Furthermore, this information has the potential to inform the development of specific antipseudomonal therapies that quench possible drug-sequestering vesicle proteins. This strategy could serve as a novel approach for combating the high-level of antimicrobial resistance in P. aeruginosa biofilms.