Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

The Synergy between Antibiotics and the Nanoparticle-Based Photodynamic Effect.

Antimicrobial resistance (AMR) is a growing global health concern, necessitating innovative strategies beyond the development of new antibiotics. Here, we employed NdYVO4:Eu3+ nanoparticles, which can persistently produce reactive oxygen species (ROS) after stopping the light, as a model of photodynamic nanoparticles and demonstrated that the photodynamic effect can serve as an adjuvant with antibiotics to effectively reduce their minimum inhibitory concentration. These preirradiated nanoparticles could penetrate the bacterial cell membrane, significantly enhancing the potency of antibiotics. We showed that the synergy effect could be attributed to disrupting crucial cellular processes by ROS, including damaging cell membrane proteins, interfering with energy supply, and inhibiting antibiotic metabolism. Our findings suggested that complementing the photodynamic effect might be a robust strategy to enhance antibiotic potency, providing an alternative antibacterial treatment paradigm.

Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosis.

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In Mtb-infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1ß secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.IMPORTANCEAddressing drug-resistant tuberculosis with existing therapies is challenging and the treatment success rate is lower when compared to drug-susceptible infection. This study demonstrates that pairing beta-lactams with ethambutol (EMB) significantly improves their efficacy against Mycobacterium tuberculosis (Mtb). The presence of EMB enhances beta-lactam access through the cell wall, which may translate into a prolonged contact between the drug and its targets at a concentration that effectively kills the pathogen. Importantly, we showed that the effects of the EMB and meropenem (MEM)/clavulanate combination were maintained intracellularly. These results are of high significance considering that the time above the minimum inhibitory concentration is the main determinant of beta-lactam efficacy. Moreover, a correlation was established between incubation with higher MEM concentrations during macrophage infection and increased IL-1ß secretion. This finding unveils a previously overlooked aspect of carbapenem repurposing against tuberculosis, as certain Mtb strains suppress the secretion of this key pro-inflammatory cytokine to evade host surveillance.

Isolation and characterization of bacteriophages against E.coli urinary tract infection and evaluating their anti-biofilm activity and antibiotic synergy.

Urinary tract infections (UTIs) by Uropathogenic Escherichia coli (UPEC) are a significant health concern, especially due to the increasing prevalence of antibiotic resistance. This study focuses on isolating and characterizing bacteriophages specific to UPEC strains isolated from UTI samples. The isolated phages were assessed for their ability to target and lyse UPEC in vitro, focusing on their efficacy in disrupting biofilms, a key virulence factor contributing to UTI recurrence and antibiotic resistance. The morphological structure observed by TEM belongs to Myoviridae, the phage exhibited icosahedral symmetry with a long non-constricting tail, the approximate measurement of the phage head was 39 nm in diameter, and the phage tail was 105.317 nm in length. One-step growth experiments showed that the latent period was approximately 20 min, followed by a rise period of 40 min, and a growth plateau was reached within 20 min and the burst size observed was 26 phages/infected bacterial cells. These phages were capable of killing cells within the biofilms, leading to a reduction in living cell counts after a single treatment. This study highlights the potential of phages to play a significant role in disrupting, inactivating, and destroying Uropathogenic Escherichia coli (UPEC) biofilms. Such findings could be instrumental in developing treatment strategies that complement antibiotics and disinfectants. The phage-antibiotic synergistic activity was compared to have the possibility to facilitate the advancement of focused and enduring alternatives to traditional antibiotic therapies for UTIs.

Assessment of the potential of phage-antibiotic synergy to induce collateral sensitivity in Salmonella Typhimurium.

This study was designed to evaluate the synergistic effect of phage and antibiotic on the induction of collateral sensitivity in Salmonella Typhimurium. The synergistic effects of Salmonella phage PBST32 combined with ciprofloxacin (CIP) against S. Typhimurium KCCM 40253 (STKCCM) were evaluated using a fractional inhibitory concentration (FIC) assay. The CIP susceptibility of STKCCM was increased when combined with PBST32, showing 16-fold decrease at 7 log PFU/mL. The combination of 1/2 × MIC of CIP and PBST32 (CIP[1/2]+PBST32) effectively inhibited the growth of STKCCM up to below the detection limit (1.3 log CFU/mL) after 12 h of incubation at 37 °C. The significant reduction in bacterial swimming motility was observed for PBST32 and CIP[1/4]+PBST32. The CIP[1/4]+PBST32 increased the fitness cost (relative fitness = 0.57) and decreased the cross-resistance to different classes of antibiotics. STKCCM treated with PBST32 alone treatment exhibited the highest coefficient of variation (90%), followed by CIP[1/4]+PBST32 (75%). These results suggest that the combination of PBST32 and CIP can be used to control bacterial pathogens.

RNase HI Depletion Strongly Potentiates Cell Killing by Rifampicin in Mycobacteria.

Multidrug-resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and they can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin, and rifampicin was increased, the latter by a striking near 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and rifampicin. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action and providing the first step in developing a new class of antimycobacterial drug.

Bacteriophages and antibiotic interactions in clinical practice: what we have learned so far.

Bacteriophages (phages) may be used as an alternative to antibiotic therapy for combating infections caused by multidrug-resistant bacteria. In the last decades, there have been studies concerning the use of phages and antibiotics separately or in combination both in animal models as well as in humans. The phenomenon of phage-antibiotic synergy, in which antibiotics may induce the production of phages by bacterial hosts has been observed. The potential mechanisms of phage and antibiotic synergy was presented in this paper. Studies of a biofilm model showed that a combination of phages with antibiotics may increase removal of bacteria and sequential treatment, consisting of phage administration followed by an antibiotic, was most effective in eliminating biofilms. In vivo studies predominantly show the phenomenon of phage and antibiotic synergy. A few studies also describe antagonism or indifference between phages and antibiotics. Recent papers regarding the application of phages and antibiotics in patients with severe bacterial infections show the effectiveness of simultaneous treatment with both antimicrobials on the clinical outcome.

PhageScore-based analysis of Acinetobacter baumannii infecting phages antibiotic interaction in liquid medium.

The growing interest in bacteriophages and antibiotics' combined use poses new challenges regarding this phenomenon's accurate description. This study aimed to apply the PhageScore methodology to assess the phage-antibiotic combination activity in liquid bacterial culture. For this purpose, previously described Acinetobacter infecting phages vB_AbaP_AGC01, Aba-1, and Aba-4 and antibiotics (gentamicin, ciprofloxacin, meropenem, norfloxacin, and fosfomycin) were used to obtain a lysis curve of bacteriophages under antibiotic pressure. The experimental data were analyzed using the Fractional Inhibitory Concentration Index (FICI) and PhageScore methodology. The results obtained by this method clearly show differences between phage lytic activity after antibiotic addition. Thus, we present the potential use of the PhageScore method as a tool for characterizing the phage antibiotic synergy in liquid culture. Further, the optimization of the PhageScore for this purpose can help compare antibiotics and their outcome on bacteriophage lytic activity.

In vitro evaluation of antibiotic synergy for carbapenem-resistant Klebsiella pneumoniae clinical isolates.

Background & objectives: The prevalence of severe infections due to carbapenem-resistant Klebsiella pneumoniae (CRKP) strains has increased worldwide. With rising resistance to polymyxins, the treatment has become challenging. Given the paucity of novel agents and limited data on combination therapy for CRKP, the present study was performed to test antibiotic combinations, for synergy against clinical isolates of CRKP. Methods: A total of 50 clinical isolates of CRKP were included. Modified carbapenem inactivation method was performed for the detection of carbapenemases. In vitro synergy testing was done for the following combinations: meropenem+colistin, imipenem+tigecycline and polymyxin B+levofloxacin. It was performed with epsilometric test and microdilution checkerboard method. The time kill assay (TKA) was used to confirm the results. The fractional inhibitory concentration was also calculated. Results: All CRKP isolates (100%) were ESBL producers and were completely resistant to amoxicillin-clavulanic acid, cefepime, cefotaxime, ceftazidime and piperacillin-tazobactam. Resistance to ciprofloxacin, amikacin and tetracycline was 96, 88 and 54 per cent, respectively. Overall, 78 (39/50) and 88 per cent (44/50) of the 50 CRKP isolates exhibited synergy by TKA for meropenem-colistin and imipenem-tigecycline, respectively. No synergy was detected for levofloxacin-polymyxin B combination. The best combination among the three was that of imipenem and tigecycline followed by meropenem-colistin. Interpretation & conclusions: Of the three combinations tested, imipenem and tigecycline followed by meropenem-colistin were found to be best. No synergy was detected for levofloxacin-polymyxin B combination.

Thrombin-Derived Peptides Potentiate the Activity of Gram-Positive-Specific Antibiotics against Gram-Negative Bacteria.

The continued rise of antibiotic resistance threatens to undermine the utility of the world's current antibiotic arsenal. This problem is particularly troubling when it comes to Gram-negative pathogens for which there are inherently fewer antibiotics available. To address this challenge, recent attention has been focused on finding compounds capable of disrupting the Gram-negative outer membrane as a means of potentiating otherwise Gram-positive-specific antibiotics. In this regard, agents capable of binding to the lipopolysaccharide (LPS) present in the Gram-negative outer membrane are of particular interest as synergists. Recently, thrombin-derived C-terminal peptides (TCPs) were reported to exhibit unique LPS-binding properties. We here describe investigations establishing the capacity of TCPs to act as synergists with the antibiotics erythromycin, rifampicin, novobiocin, and vancomycin against multiple Gram-negative strains including polymyxin-resistant clinical isolates. We further assessed the structural features most important for the observed synergy and characterized the outer membrane permeabilizing activity of the most potent synergists. Our investigations highlight the potential for such peptides in expanding the therapeutic range of antibiotics typically only used to treat Gram-positive infections.