Fidaxomicin 200mg tablets

PURPOSE: To compare the efficacy, recurrence rate, adverse event rate and mortality of fidaxomicin compared with vancomycin in treating different types of Clostridium difficile infection (CDI). METHODS: A systematic search was conducted on PubMed, Embase, Web of Science, Cochrane Library and clinical trial registration databases for research on fidaxomicin versus vancomycin in the treatment of CDI and the retrieval period extended from the establishment of the database to July 22, 2022. A total of 15 studies were included, including 8 RCTs and 7 retrospective cohort studies. RESULTS: Results showed that there was no significant difference in the overall efficacy of the treatment between fidaxomicin and vancomycin, and results in the subgroups of CDI hypervirulent strains and recurrent CDI were obtained, but vancomycin was more effective than fidaxomicin in the treatment of severe CDI (RR = 0.94, 95% CI: 0.90-0.98, P < .01). Results showed that fidaxomicin is superior to vancomycin in terms of 40-day recurrence rate (RR = 0.52, 95% CI: 0.38-0.70, P < .01), 60-day recurrence rate (RR = 0.38, 95% CI: 0.21-0.69, P < .01) and 90-day recurrence rate (RR = 0.62, 95% CI: 0.50-0.77, P < .01). For the recurrence rate of the treatment in CDI hypervirulent strains, severe CDI and recurrent CDI, there was no significant difference between the 2 groups. In addition, there was no significant difference in the incidence of clinical adverse reactions, and same outcomes appeared in all-cause mortality at 40-day, severe CDI and recurrent CDI, but fidaxomicin was superior to vancomycin in all-cause mortality over 60-day (RR = 0.57, 95% CI: 0.34-0.96, P = .03). CONCLUSION: There were no significant differences between fidaxomicin and vancomycin in the treatment of CDI in therapeutic effectiveness and adverse reactions, while fidaxomicin was superior to vancomycin in terms of recurrence rate and long-term mortality, and vancomycin is more effective in treating severe CDI.

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The reviewer highlighted several critical concerns regarding scientific rigor, including a very small sample size (n=3), the exclusive use of male mice, and the lack of whole-genome sequencing to ensure no other mutations influenced the results.

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Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket. https://doi.org/10.7554/eLife.34823.001 eLife digest Tuberculosis (TB) is an infectious disease that affects over ten million people every year. The Mycobacterium tuberculosis bacteria that cause the disease spread through the air from one person to another and mainly infect the lungs. Although curable, TB is difficult to eradicate because it is remarkably widespread, with one third of the world’s population estimated to carry the bacteria. Treatment for TB involves a mix of antibiotics that should be taken for several months to a year. The number of multidrug-resistant TB cases, where the infection is not treatable by the common cocktail of antibiotics, is rapidly increasing. There is therefore a need to discover new drugs that can kill the M. tuberculosis bacteria. An antibiotic called fidaxomicin is used to treat intestinal infections. Although it can kill Mycobacterium tuberculosis cells in culture, it is not absorbed from the intestines to the blood and thus cannot reach the lungs to kill the bacteria. It may be possible to change the structure of the drug so that it can enter the bloodstream. Before this can be done, researchers need to understand exactly how fidaxomicin kills the bacteria so that they know which parts of the drug they can alter without making it less effective. Fidaxomicin kills bacterial cells by binding to an enzyme called RNA polymerase. The antibiotic prevents the enzyme from reading and ‘transcribing’ DNA to form molecules that are essential for life. To learn more about how fidaxomicin has this effect, Boyaci, Chen et al. used cryo-electron microscopy to look at structures of the M. tuberculosis RNA polymerase in different states, including when it was bound to fidaxomicin. The structures reveal the chemical details of the interactions between the RNA polymerase and the antibiotic. The two molecules bind to each other through a region of the RNA polymerase that is unique to M. tuberculosis and closely related bacteria. Fidaxomicin acts like a doorstop to jam the RNA polymerase in an open state that cannot bind to DNA and transcribe genes. Medicinal chemists could now build on these findings to develop new drugs that might treat TB, either by modifying fidaxomicin or designing new antibiotics that bind to the same region of the RNA polymerase. Because the fidaxomicin-binding region of the RNA polymerase is specific to M. tuberculosis new antibiotics could be tailored towards the bacteria that have a minimal effect on a patient’s normal gut bacteria. https://doi.org/10.7554/eLife.34823.002 Introduction The bacterial RNA polymerase (RNAP) is a proven target for antibiotics. The rifamycin (Rif) class of antibiotics, which inhibit RNAP function, is a lynchpin of modern tuberculosis (TB) treatment (Chakraborty and Rhee, 2015). TB, caused by the infectious agent Mycobacterium tuberculosis (Mtb), is responsible for almost 2 million deaths a year. It is estimated that one third of the world is infected. Mortality from TB is increasing, partly due to the emergence of strains resistant to Rifs (RifR) (Zumla et al., 2015). Hence, additional antibiotics against RifR Mtb are needed. Fidaxomicin (Fdx; also known as Dificimicin, lipiarmycin, OPT-80, PAR-101, or tiacumicin), an antimicrobial in clinical use against Clostridium difficile (Cdf) infection (Venugopal and Johnson, 2012), functions by inhibiting the bacterial RNAP (Talpaert et al., 1975). Fdx targets the RNAP 'switch region', a determinant for RNAP inhibition that is distinct from the Rif binding pocket (Srivastava et al., 2011), and Fdx does not exhibit cross-resistance with Rif (Gualtieri et al., 2009, 2006; Kurabachew et al., 2008; O'Neill et al., 2000). The switch region sits at the base of the mobile RNAP clamp domain and, like a hinge, controls motions of the clamp crucial for DNA loading into the RNAP active-site cleft and maintaining the melted DNA in the channel (Chakraborty et al., 2012; Feklistov et al., 2017). Fdx is a narrow spectrum antibiotic that inhibits Gram-positive anaerobes and mycobacteria (including Mtb) much more potently than Gram-negative bacteria (Kurabachew et al., 2008; Srivastava et al., 2011), but the clinical use of Fdx is limited to intestinal infections due to poor bioavailability (Venugopal and Johnson, 2012). Addressing this limitation requires understanding the structural and mechanistic basis for Fdx inhibition, which is heretofore unknown. Here, we used single-particle cryo-electron microscopy (cryo-EM) to determine structures of Mtb transcription initiation complexes in three distinct conformational states, including a complex with Fdx at an overall resolution of 3.4 Å. The results define the molecular interactions of Mtb RNAP with Fdx as well as the mechanistic basis of inhibition, and establish that RbpA, an Actinobacteria-specific general transcription factor (GTF), is crucial to the sensitivity of Mtb to Fdx. Results Fdx potently inhibits mycobacterial TICs in vitro Fdx has potent inhibitory activity against multi-drug-resistant Mtb cells and the in vivo target is the RNAP (Kurabachew et al., 2008). To our knowledge, the in vitro activity of Fdx against mycobacterial RNAPs has not been reported. RbpA, essential in Mtb, is a component of transcription initiation complexes (TICs) that tightly binds the primary promoter specificity σA subunit of the RNAP holoenzyme (holo) (Bortoluzzi et al., 2013; Forti et al., 2011; Hubin et al., 2017a, 2015; Tabib-Salazar et al., 2013). We therefore compared Fdx inhibition of mycobacterial RNAPs containing core RNAP combined with σA (σA-holo) and RbpA with inhibition of Escherichia coli (Eco) σ70-holo using a quantitative abortive initiation assay (Davis et al., 2015). Fdx inhibited Mtb and M. smegmatis (Msm) transcription at sub-μM concentrations, whereas inhibition of an Mtb TIC containing Fdx-resistant (FdxR) RNAP (βQ1054H) (Kurabachew et al., 2008) required a nearly two orders of magnitude higher concentration of Fdx. Eco RNAP was inhibited even less effectively by another order of magnitude (Figure 1A, Figure 1—figure supplement 1A). Figure 1 with 3 supplements see all Download asset Open asset Structure of an Mtb RbpA/TIC with Fdx at 3.4 Å resolution. (A) Fdx inhibits mycobacterial RbpA/σA-holo transcription greater than 250-fold more effectively than Ecoσ70-holo in in vitro abortive initiation assays. The error bars denote the standard error from a minimum of three experiments (for some points, the error bars are smaller than the width of the point and are not shown). (B) Chemical structure of Fdx (Serra et al., 2017). (C) Synthetic us-fork promoter fragment used for cryo-EM experiments. The DNA sequence is derived from the full con promoter (Gaal et al., 2001). The nontemplate-strand DNA (top strand) is colored light gray; the template-strand DNA (bottom strand), dark grey. The −35 and −10 elements are shaded yellow. The extended −10 (Keilty and Rosenberg, 1987) is colored violet. (D) The 3.4 Å resolution cryo-EM density map of the Fdx/RbpA/σA-holo/us-fork complex is rendered as a transparent surface colored as labeled. Superimposed is the final refined model; proteins are shown as a backbone ribbon, Fdx and the nucleic acids are shown in stick format. (E) Views of the cryo-EM map colored by local resolution based on blocres calculation (Cardone et al., 2013). The left view shows the entire map, while the middle view shows a cross-section of the map sliced at the level of the Fdx binding pocket. The boxed region is magnified on the right. Density for the Fdx molecule is outlined in red. https://doi.org/10.7554/eLife.34823.003 Cryo-EM structure of the Fdx/RbpA/σA-holo complex We used single-particle cryo-EM to examine the complex of Mtb RbpA/σA-holo with and without Fdx (Figure 1B). Preliminary analyses revealed that the particles were prone to oligomerization, which was reduced upon addition of an upstream-fork (us-fork) junction promoter DNA fragment (Figure 1C). We sorted nearly 600,000 cryo-EM images of individual particles into two distinct classes, each arising from approximately half of the particles (Figure 1—figure supplement 2). The first class comprised Mtb RbpA/σA-holo with one us-fork promoter fragment and bound to Fdx. The cryo-EM density map was computed to a nominal resolution of 3.4 Å (Figure 1D, Figure 1—figure supplement 3, Supplementary file 1). The us-fork promoter fragment was bound outside the RNAP active site cleft, as expected, with the −35 and −10 promoter elements engaged with the σA4 and σA2 domains, respectively (Figure 1D). Local resolution calculations (Cardone et al., 2013) indicated that the central core of the structure, including the Fdx binding determinant and the bound Fdx, was determined to 2.9–3.4 Å resolution (Figure 1E). Cryo-EM structure of a Mtb RPo mimic The second class comprised Mtb RbpA/σA-holo bound to two us-fork promoter fragments but without Fdx to a nominal resolution of 3.3 Å (Figure 2A, Figure 2—figure supplement 1, Supplementary file 1). One us-fork promoter fragment bound upstream from the RNAP active site cleft as in the previous class, but a second us-fork promoter fragment bound the RNAP downstream duplex DNA binding channel, with the 5-nucleotide 3'-overhang (Figure 1C) engaged with the RNAP active site (as the template strand) like previously characterized 3'-tailed templates (Gnatt et al., 2001; Kadesch and Chamberlin, 1982). Local resolution calculations (Cardone et al., 2013) indicated that the central core of the structure was determined to between 2.8–3.2 Å resolution (Figure 2B). The overall conformation of this protein complex and its engagement with the upstream and downstream DNA fragments was very similar to the crystal structure of a full Msm open promoter complex (RPo) (Hubin et al., 2017b) with one exception (see below). We will therefore call this complex an Mtb RbpA/RPo mimic. Figure 2 with 1 supplement see all Download asset Open asset Structure of an Mtb RbpA/RPo mimic at 3.3 Å resolution. (A) The 3.3 Å resolution cryo-EM density map of the RbpA/σA-holo/(us-fork)2 complex (RbpA/RPo mimic) is rendered as a transparent surface colored as labeled. Superimposed is the final refined model; proteins are shown as a backbone ribbon, nucleic acids are shown in stick format. (B) Views of the Mtb RbpA/RPo mimic cryo-EM map colored by local resolution based on blocres calculation (Cardone et al., 2013). The left view shows the entire map, while the middle view shows a cross-section of the map sliced at the level of the RbpANTT. The boxed region is magnified on the right. Density for the RbpANTT is outlined in red. https://doi.org/10.7554/eLife.34823.007 The RbpA N-terminal tail invades the RNAP active site cleft RbpA comprises four structural elements, the N-terminal tail (NTT), the core domain (CD), the basic linker, and the sigma interacting domain (SID) (Bortoluzzi et al., 2013; Hubin et al., 2017a; Tabib-Salazar et al., 2013). Our previous crystal structures of Msm TICs containing RbpA showed that the RbpASID interacts with the σA2 domain, the RbpABL establishes contacts with the promoter DNA phosphate backbone just upstream of the −10 element, and the RbpACD interacts with the RNAP β' Zinc-Binding-Domain (ZBD) (Hubin et al., 2017a, 2017b). Density for the RbpANTT (RbpA residues 1–25) was never observed in the crystal structures and was presumed to be disordered. In striking contrast to the crystal structures, both cryo-EM structures reveal density for the RbpANTT, which unexpectedly threads into the RNAP active site cleft between the ZBD and σA4 domains and snakes through a narrow channel towards the RNAP active site Mg2+ (Figure 3). On its path, conserved residues of the RbpANTT interact with conserved residues of the σ-finger (σ3.2-linker) on one wall of the channel, and with conserved residues of the ZBD and β'lid on the other wall (Figure 3C). Figure 3 Download asset Open asset The RbpANTT interacts with conserved structural elements in the RNAP active site cleft. (A) An overview of the RbpA/RPo structure is shown as a color-coded molecular surface (color-coding denoted in the key) except the β flap and σA4 domain are shown as backbone worms, revealing the RbpANTT (magenta) underneath. The DNA fragments are not shown. The boxed region is magnified in panel (B). (B) Magnified view of the boxed region from panel (A). The RbpANTT is shown in stick format with a transparent molecular surface. Conserved RNAP structural elements that interact with the RbpANTT are highlighted (βSw3, β'ZBD, β'Zipper, β'Lid, and σ-finger). (C) Further magnified view showing the cryo-EM density (blue mesh) with the superimposed model. Conserved residues of the RbpANTT are labeled, along with conserved residues of the β'ZBD, β'Lid, and σ-finger that interact with the RbpANTT. https://doi.org/10.7554/eLife.34823.009 The most N-terminal RbpA residues visible in the cryo-EM structures (A2 in the Fdx complex, R4 in the RPo) sit near the tip of the σ-finger where it makes its closest approach to the RNAP active site, too far (25 Å) to play a direct role in RNAP catalytic activity or substrate binding. The σ-finger plays an indirect role in transcription initiation, stimulating de novo phosphodiester bond formation by helping to position the t-strand DNA (Kulbachinskiy and Mustaev, 2006; Zhang et al., 2012). The σ-finger is also a major determinant of abortive initiation, playing a direct role in initiation and promoter escape by physically blocking the path of the elongating RNA transcript before σ release (Cashel et al., 2003; Murakami et al., 2002). The intimate association of the RbpANTT with the σ-finger (Figure 3C) suggests that the RbpANTT also plays a role in these processes of Mtb RNAP initiation. This is consistent with our findings that the RbpANTT does not strongly affect RPo formation but plays a significant role in in vivo gene expression in Msm (Hubin et al., 2017a). This location of the RbpANTT explains the high Fdx sensitivity of Mtb RNAP (see below). Fdx interacts with RNAP, σA, and RbpA The reconstruction from the Fdx-bound class (Figure 1D) reveals unambiguous density for Fdx (Figure 4A) and defines Fdx-interacting residues from four protein components of the complex, β, β', σA, and RbpA, including six water molecules, four of which mediate Fdx/RNAP interactions (Figure 4A,B). Fdx binding to the TIC buries a large accessible surface area of 4,800 Å2 (β, 2,100 Å2; β', 2,000 Å2; σA, 300 Å2; RbpA, 330 Å2). Fdx forms direct hydrogen bonds with nine residues (βQ1054, βD1094, βT1096, βK1101, β'R84, β'K86, β'R89, β'E323, and β'R412) and water-mediated interactions with four (β'R89, β'D404, β'Q415, and RbpA-E17). Notably, the Fdx/RNAP interaction is stabilized by two cation-π interactions between β'R84 and the aromatic ring of the Fdx homodichloroorsellinic acid moiety (Figure 1B) and β'R89 and the conjugated double-bond system centered between C4 and C5 of the macrolide core (Figures 1B and 4A,B). Fdx interacts with residues from eight distinct structural elements (Lane and Darst, 2010) of the initiation complex (βSw3, βSw4, β residues belonging to the clamp, β'ZBD, β'lid, β'Sw2, the σ-finger, and the RbpaNTT (Figure 4A,B). Figure 4 Download asset Open asset Structural basis for Fdx inhibition of Mtb transcription and the role of the RbpANTT. (A) (left) Overview of the Fdx/RbpA/σA-holo/us-fork structure, shown as a molecular surface (the DNA is not shown). The boxed region is magnified on the right. (right) Magnified view of the Fdx binding pocket at the same orientation as the boxed region on the left. Proteins are shown as α-carbon backbone worms. Residues that interact with Fdx are shown in stick format. Fdx is shown in stick format with green carbon atoms. Water molecules are shown as small pink spheres. Hydrogen-bonds are indicated by dashed gray lines. Cation-π interactions (between β'R84 and the aromatic ring of the Fdx homodichloroorsellinic acid moiety and β'R89 and the conjugated double-bond system centered between C4 and C5 of the macrolide core) are represented by red dashed lines. (B) Schematic summary of the Fdx contacts with σA-holo and RbpA. Fdx is shown in stick format with green carbon atoms. Thin dashed lines represent van der Waals contacts (≤4.5 Å), thick red lines represent hydrogen bonds (<4 Å). The thin dashed red lines denote cation-π interactions. (C) The RbpANTT is required for optimal inhibition of Mtb transcription by Fdx in in vitro abortive initiation assays. The error bars denote the standard error from a minimum of three experiments (for some points, the error bars are smaller than the width of the point and are not shown). (D) Zone of inhibition assays with Msm cells show that loss of the RbpA-NTT (RbpAΔNTT) leads to loss of Fdx sensitivity in vivo. https://doi.org/10.7554/eLife.34823.010 Amino-acid substitutions conferring FdxR have been identified in RNAP β or β' subunits from Bacillus subtilis (Gualtieri et al., 2006), Cdf (Kuehne et al., 2017), Enterococcus faecalis (Gualtieri et al., 2009), and Mtb (Kurabachew et al., 2008), corresponding to Mtb RNAP β residues Q1054 (Sw3), V1100 and V1123 (clamp), and β' residues R89 (ZBD), P326 (lid), and R412 (Sw2). The structure shows that each of these residues makes direct interactions with Fdx (Figure 4A,B). All five chemical moieties of Fdx (Figure 1B) interact with at least one RNAP residue that confers FdxR when mutated (Figure 4B), suggesting that each moiety may be important for Fdx action. The RbpANTT is critical for Fdx potency against mycobacterial RNAP in vitro and in vivo In addition to the β and β' subunits, Fdx interacts with residues of the σ-finger (D424 and V445; Figure 4A,B). Finally and unexpectedly, Fdx contacts residues from the RbpANTT (Figure 4A,B). To test the functional importance of the RpbANTT for Fdx inhibition in vitro, we compared Fdx inhibition of MtbσA-holo with either RbpA or RbpA with the NTT truncated (RbpAΔNTT) in the abortive initiation assay (Figure 1—figure supplement 1B). Truncation of the RbpA-NTT caused a 35-fold increase in resistance to Fdx (Figure 4C). RbpA is essential in Mtb and Msm, but strains carrying are (Hubin et al., to test the role of the RbpANTT in Fdx inhibition of Msm We of inhibition assays on two Msm strains that are except one RbpA and the other (Hubin et al., 2017a). The Msm on approximately the as the Msm to reach the the was less to Fdx (Figure with to Fdx not inhibition with but inhibition were with Fdx, the inhibition for was smaller than for a protein inhibition for the and We that the essential role of RbpA in Mtb transcription is to the high sensitivity of Mtb cells to Fdx. Fdx an conformation The RNAP switch are to as the mobile clamp domain to the of the RNAP (Gnatt et al., 2001; and Darst, RNAP and bind and and a conformation of the RNAP et al., et al., 2008). The Fdx binding determinant does not the for these other but the Fdx interactions with the and (Figure that Fdx may the clamp conformation as To understand the role of Fdx in clamp without the of DNA binding in the RNAP active site cleft, we determined cryo-EM structures of Mtb RbpA/σA-holo without with Fdx and without Fdx. Although the particles in the cryo-EM of Mtb RbpA/σA-holo were prone to oligomerization, we used to particles and determined of Mtb RbpA/σA-holo without DNA and with Fdx Å resolution from Figure Figure supplement and without Fdx Å resolution from Figure Figure supplement The cryo-EM density were of to the bound antibiotic in the Fdx complex (Figure supplement and to determine the domain (including the clamp by (Figure we were to the RNAP conformational states from four complexes of the same RNAP in the of crystal (Figure Figure with 1 supplement see all Download asset Open asset of Fdx inhibition of bacterial (A) Cryo-EM density and superimposed refined for Mtb RbpA/σA-holo Å and Mtb Fdx/RbpA/σA-holo Å (B) RNAP clamp conformational for four cryo-EM structures determined in this The RbpA/RPo (Figure structure was used as a to the other structures α-carbon of the structural core file revealing a common core RNAP structure as a gray molecular but with large in the clamp The clamp are shown as backbone with and color-coded clamp of clamp of open clamp of open clamp of The clamp conformational can be characterized as about a to the The of clamp for the different structures are shown to the RPo clamp, (C) The core RNAP from the 3.4 Å resolution Fdx/RbpA/σA-holo/us-fork structure is shown as a gray molecular surface but with the open clamp colored The structure is sliced at the level of the Fdx binding pocket (the bound Fdx is shown in The boxed region is magnified showing the of the Fdx molecule in a narrow between the clamp and the of the (D) The core RNAP from the 3.3 Å resolution RbpA/RPo structure is shown as a gray molecular surface but with the clamp colored The structure is sliced at the level of the Fdx binding pocket. Fdx, from the structure shown in is shown in The boxed region is magnified Fdx cannot bind to RNAP with a clamp because clamp the Fdx binding site. is required for initiation and of the transcription et al., and also for binding of nucleic acids in the RNAP cleft. The four structures were superimposed by the structural core file the subunit and highly conserved β and in or near the active that have not been observed to significant conformational in of RNAP the RPo structure (Figure as a the structures superimposed with Å over at least α-carbon of the structural core but Å for of the clamp file large of the clamp with to the of the RNAP in the different of the structures revealed that the clamp conformational could be characterized as about a common (Figure a clamp of to the RPo structure Figure the RbpA/σA-holo clamp is open by about Figure Because this complex is not interacting with other that might be to alter the clamp conformation as Fdx or we will to this as the clamp The two Fdx-bound with or without us-fork show of the clamp and and red in Figure Fdx acts like a doorstop to the conformation In the structure (Figure Fdx binds in a narrow between the open clamp and the of the RNAP (Figure of the RPo structure reveals that clamp the Fdx binding pocket (Figure Fdx can bind to the conformation of We thus that Fdx acts like a binding and the Discussion Fdx inhibits RNAP by an conformation play important in the transcription of the clamp and the role of the switch as were first by crystal structures of free RNAPs et al., 2001; Zhang et al., with the crystal structure of an complex containing template DNA and RNA transcript (Gnatt et al., 2001). of the downstream duplex DNA and in the RNAP active-site cleft was to the clamp the nucleic explaining the high of the transcription crystal structures have the that complexes of RNAP with nucleic either RPo et al., 2015; Hubin et al., and or complexes (Gnatt et al., 2001; et al., et al., with the of crystal on clamp cannot be of clamp by (Chakraborty et al., 2012), and more in cryo-EM structures et al., et al., 2015; et al., et al., the of crystal have the between clamp and motions have also been shown to play a critical role in the of promoter to form the transcription RPo formation et al., 2017). the of an RNAP conformation by Fdx in cryo-EM (Figure suggests that Fdx inhibits transcription initiation by preventing clamp motions required for RPo or by not RNAP to form complexes with nucleic or both (Figure results are consistent with mechanistic analyses of et al., 2010) and et al., showing that Fdx promoter at an but RNAP a template the that Fdx the clamp from consistent with our structural Our results establish the molecular details of Fdx interactions with the bacterial RNAP (Figure and a of for Fdx (Figure the essential RbpA is responsible for the high sensitivity of mycobacterial RNAP to Fdx both in vitro (Figure and in vivo (Figure This new a structural platform for the development of antimicrobials that target the Fdx binding determinant and the need to define of drug leads using states, in this using cryo-EM with the RbpA/σA-holo complex to guide development of effective Mtb Materials and methods or or smegmatis Msm

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BACKGROUND: There have been several recent reports of Clostridioides difficile infection (CDI) due to isolates with reduced fidaxomicin susceptibility (minimum inhibitory concentration [MIC] ≥ 2 µg/mL). However, the clinical implications are uncertain because fidaxomicin achieves high concentrations in the intestinal tract. METHODS: In an acute care hospital, we conducted a 3-year cohort study of patients with CDI to determine the frequency of infection with isolates with reduced fidaxomicin susceptibility and the impact on response to fidaxomicin treatment. Stool specimens were cultured for C. difficile, and susceptibility testing was performed using agar dilution. Whole-genome sequencing was used to identify mutations associated with reduced fidaxomicin susceptibility and to determine relatedness of isolates. For genomically related susceptible and reduced susceptibility isolates from the same patient, we compared rates of growth, sporulation, and toxin production. RESULTS: Of 108 fidaxomicin-treated patients, 6 (5.6%) were infected with isolates that possessed reduced fidaxomicin susceptibility (MICs 8-32 µg/mL), including 3 with initially susceptible isolates followed by clinical failure with subsequent recovery of genomically related isolates with reduced susceptibility. Isolates with reduced fidaxomicin susceptibility harbored mutations in RNA polymerase associated with reduced susceptibility and exhibited reduced toxin production, and 20% to 40% of isolates tested had reduced growth and/or sporulation in comparison with susceptible isolates. Three patients were infected with genomically indistinguishable ribotype 097 isolates with reduced fidaxomicin susceptibility. CONCLUSIONS: Our findings highlight the potential for the emergence on therapy of clinically relevant reduced fidaxomicin susceptibility in C. difficile and its spread via transmission to other patients.

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and the broad spectrum bacteriocin nisin in a complex community.

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BACKGROUND: Antibiotics are currently the primary treatment of Clostridioides difficile (C. difficile) infection. Yet, due to rapid development of resistance and high recurrences rates, there is an unmet need for new antimicrobials for C. difficile infections. This study assessed the in vitro susceptibility of clinical isolates from Israel to two recently developed antibiotics, ridinilazole (RDZ) and ibezapolstat (IBZ), and to standard-of-care antibiotics. METHODS: C. difficile isolates (n = 313) recovered from patients at both community and hospital medical centers across Israel, were typed to different sequence types (ST) by multi-locus sequencing typing (MLST). Susceptibility to metronidazole (MTZ) and vancomycin (VAN) was determined using the gradient strip test (Etest). Susceptibility to fidaxomicin (FDX), RDZ and IBZ was determined by agar dilution. RESULTS: of 4 mg/L. No significant differences were noted in IBZ MIC of different strains. CONCLUSIONS: RDZ and IBZ demonstrated potent in vitro activity against 313 C. difficile isolates belonging to different STs. These two antimicrobials may serve as effective agents for C. difficile infection.

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Abstract Introduction: Clostridioides difficile infection (CDI) is a common nosocomial infection and is associated with a high healthcare burden due to high rates of recurrence. In 2021 the IDSA/SHEA guideline update recommended fidaxomicin (FDX) as first-line therapy. Our medical center updated our institutional guidelines to follow these recommendations, prioritizing FDX use among patients at high risk for recurrent CDI (rCDI). Methods: This pre- post- quasi-experimental study included patients with a presumptive diagnosis of CDI at risk for recurrence (age &gt;/= 65 years, immunocompromised, severe CDI) that received vancomycin (VAN) or FDX between October 2019 to October 2022. Patients who received bezlotoxumab, had fulminant CDI, or received &lt;10 days of the same antibiotic for their full treatment course were excluded. Patients were evaluated for rCDI within 8 weeks of completion of therapy, subsequent episodes of CDI within 12 months, and CDI-related admissions within 30 days. Results: Of 397 CDI regimens evaluated, 196 received VAN and 201 received FDX. Rates of rCDI (9.2% vs 10%, P = 0.86), subsequent CDI within 12 months of therapy completion of therapy (19.4% vs 26%, P = 0.12) and 30-day CDI-related readmissions (3% vs 4.5%, P = 0.6) were similar between patients who received VAN versus FDX. Conclusion: Outcomes were similar between patients treated with FDX and VAN for the treatment of CDI among those at high risk for rCDI, using our outlined criteria. Although we observed a trend toward lower rates of rCDI among immunocompromised patients, this finding was not significant. Further investigation is needed to determine which patients with CDI may benefit from FDX.

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Abstract Objective: Compare the real-world impact of fidaxomicin (FDX) and vancomycin (VAN) on Clostridioides difficile infection (CDI) recurrence in a high-risk patient population. Design: A retrospective, matched-cohort study evaluating hospitalized patients with CDI from January 1, 2016, to November 1, 2022, within a tertiary academic medical center. Patients: Adult patients with at least 1 prior CDI case who received either FDX or VAN for non-fulminant CDI while admitted, and had at least 1 additional risk factor for recurrence. Risk factors included age &gt;70, solid organ or bone marrow transplant recipients, broad-spectrum antibiotic use within 30 days, or receipt of chemotherapy/immune-modulating agents within 30 days of admission. FDX and VAN patients were matched according to risk factors. Results: A total of 415 patient admissions were identified. After the exclusion of 92 patients for fulminant CDI, diarrhea from another cause, or use of VAN taper therapy, and 15 unmatched patients, 308 patient admissions were included (68 FDX and 240 VAN patients). There were no significant differences in 4-week recurrence (26% vs 23%; OR 1.1; P = .51), 90-day CDI readmission (29% vs 23%; P = .65), or 90-day all-cause readmission (54% vs 53%; P = .91). There was a significant 17% decrease in 90-day mortality associated with the use of FDX (OR .3; P = .04). Conclusions: In a real-world high-risk patient population, the use of FDX compared to oral VAN did not result in decreased CDI recurrence within 4 weeks or fewer hospital readmissions within 90 days. Further research is needed to better assess the value of FDX in this patient population.

Abstract licence: CC BY