HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are potent inhibitors of prokaryotic RNA polymerase (RNAP) used to treat tuberculosis and other bacterial infections. Although resistance arises in the clinic principally through mutations in RNAP, many bacteria possess highly specific enzyme-mediated resistance mechanisms that modify and inactivate rifamycins. The expression of these enzymes is controlled by a 19-bp cis-acting rifamycin-associated element (RAE). Guided by the presence of RAE sequences, we identify a helicase-like protein, HelR, in Streptomyces venezuelae that confers broad-spectrum rifamycin resistance. We show that HelR also promotes tolerance to rifamycins, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP and rescues transcription inhibition by displacing rifamycins from RNAP, thereby providing resistance by target protection . Furthermore, HelRs are broadly distributed in Actinobacteria, including several opportunistic Mycobacterial pathogens, offering yet another challenge for developing new rifamycin antibiotics.

The Collapse of Infectious Disease Diagnoses Commonly Due to Communicable Respiratory Pathogens During the Coronavirus Disease 2019 Pandemic: A Time Series and Hierarchical Clustering Analysis.

Background: Nonpharmaceutical interventions such as physical distancing and mandatory masking were adopted in many jurisdictions during the coronavirus disease 2019 pandemic to decrease spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined the effects of these interventions on incidence of healthcare utilization for other infectious diseases. Methods: Using a healthcare administrative dataset, we employed an interrupted time series analysis to measure changes in healthcare visits for various infectious diseases across the province of Ontario, Canada, from January 2017 to December 2020. We used a hierarchical clustering algorithm to group diagnoses that demonstrated similar patterns of change through the pandemic months. Results: We found that visits for infectious diseases commonly caused by communicable respiratory pathogens (eg, acute bronchitis, acute sinusitis) formed distinct clusters from diagnoses that often originate from pathogens derived from the patient's own flora (eg, urinary tract infection, cellulitis). Moreover, infectious diagnoses commonly arising from communicable respiratory pathogens (hierarchical cluster 1: highly impacted diagnoses) were significantly decreased, with a rate ratio (RR) of 0.35 (95% confidence interval [CI], .30-.40; P < .001) after the introduction of public health interventions in April-December 2020, whereas infections typically arising from the patient's own flora (hierarchical cluster 3: minimally impacted diagnoses) did not demonstrate a sustained change in incidence (RR, 0.95 [95% CI, .90-1.01]; P = .085). Conclusions: Public health measures to curtail the incidence of SARS-CoV-2 were widely effective against other communicable respiratory infectious diseases with similar modes of transmission but had little effect on infectious diseases not strongly dependent on person-to-person transmission.

The Enzymes of the Rifamycin Antibiotic Resistome.

Rifamycin antibiotics include the WHO essential medicines rifampin, rifabutin, and rifapentine. These are semisynthetic derivatives of the natural product rifamycins, originally isolated from the soil bacterium Amycolatopsis rifamycinica. These antibiotics are primarily used to treat mycobacterial infections, including tuberculosis. Rifamycins act by binding to the ß-subunit of bacterial RNA polymerase, inhibiting transcription, which results in cell death. These antibiotics consist of a naphthalene core spanned by a polyketide ansa bridge. This structure presents a unique 3D configuration that engages RNA polymerase through a series of hydrogen bonds between hydroxyl groups linked to the naphthalene core and C21 and C23 of the ansa bridge. This binding occurs not in the enzyme active site where template-directed RNA synthesis occurs but instead in the RNA exit tunnel, thereby blocking productive formation of full-length RNA. In their clinical use to treat tuberculosis, resistance to rifamycin antibiotics arises principally from point mutations in RNA polymerase that decrease the antibiotic's affinity for the binding site in the RNA exit tunnel. In contrast, the rifamycin resistome of environmental mycobacteria and actinomycetes is much richer and diverse. In these organisms, rifamycin resistance includes many different enzymatic mechanisms that modify and alter the antibiotic directly, thereby inactivating it. These enzymes include ADP ribosyltransferases, glycosyltransferases, phosphotransferases, and monooxygenases.ADP ribosyltransferases catalyze group transfer of ADP ribose from the cofactor NAD+, which is more commonly deployed for metabolic redox reactions. ADP ribose is transferred to the hydroxyl linked to C23 of the antibiotic, thereby sterically blocking productive interaction with RNA polymerase. Like ADP ribosyltransferases, rifamycin glycosyl transferases also modify the hydroxyl of position C23 of rifamycins, transferring a glucose moiety from the donor molecule UDP-glucose. Unlike other antibiotic resistance kinases that transfer the γ-phosphate of ATP to inactivate antibiotics such as aminoglycosides or macrolides, rifamycin phosphotransferases are ATP-dependent dikinases. These enzymes transfer the ß-phosphate of ATP to the C21 hydroxyl of the rifamycin ansa bridge. The result is modification of a critical RNA polymerase binding group that blocks productive complex formation. On the other hand, rifamycin monooxygenases are FAD-dependent enzymes that hydroxylate the naphthoquinone core. The result of this modification is untethering of the ansa chain from the naphthyl moiety, disrupting the essential 3D shape necessary for productive RNA polymerase binding and inhibition that leads to cell death.All of these enzymes have homologues in bacterial metabolism that either are their direct precursors or share common ancestors to the resistance enzyme. The diversity of these resistance mechanisms, often redundant in individual bacterial isolates, speaks to the importance of protecting RNA polymerase from these compounds and validates this enzyme as a critical antibiotic target.

Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action.

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.

Potentiation of Tobramycin by Silver Nanoparticles against Pseudomonas aeruginosa 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 Pseudomonas aeruginosa 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 P. aeruginosa 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 P. aeruginosain vitro and may reveal a potential role for AgNP/antibiotic combinations in treating patients with chronic infections in a strain-specific manner.

Lessons from the Environmental Antibiotic Resistome.

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.

Tracking the Dynamic Relationship between Cellular Systems and Extracellular Subproteomes in Pseudomonas aeruginosa Biofilms.

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.

Antimicrobial targets localize to the extracellular vesicle-associated proteome of Pseudomonas aeruginosa grown in a biofilm.

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.