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International Journal of Antimicrobial... Feb 2024Bacterial multi-drug resistance has become a concern worldwide, especially after the emergence of carbapenemases. Adjuvants with antibacterial potentiation activity can...
Bacterial multi-drug resistance has become a concern worldwide, especially after the emergence of carbapenemases. Adjuvants with antibacterial potentiation activity can resensitise drug-resistant strains to carbapenems. However, only a few adjuvants with antibacterial potentiation activity are currently available in clinical practice. Here, we first docked the library containing more than 30,000 small molecules to carbapenemases including Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-5 (NDM-5), through in silico virtual screening to obtain lead compounds against carbapenemase-producing Enterobacterales. Meanwhile, the in vitro antibacterial potentiation assays revealed that ibandronate, azacytidine, ribostamycin sulfate and cidofovir exhibited synergistic or additive activity in the presence of meropenem, with good biocompatibility based on red blood cell hemolysis and cell viability tests. Furthermore, the combination of meropenem and azacytidine showed high efficacy in a mouse sepsis model infected with an NDM-5-producing clinical strain, with a 100% survival rate, decreased bacterial burden and alleviated pathological deterioration. These results suggest that the virtual screening is a promising strategy to identify new antibiotic adjuvants targeting carbapenemase-producing Enterobacterales.
Topics: Animals; Mice; Meropenem; Klebsiella pneumoniae; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Azacitidine; Microbial Sensitivity Tests
PubMed: 38159889
DOI: 10.1016/j.ijantimicag.2023.107076 -
Frontiers in Microbiology 2023species have been identified as agents responsible for various diseases in both humans and animals. Multidrug-resistant strains pose a significant public health threat...
BACKGROUND
species have been identified as agents responsible for various diseases in both humans and animals. Multidrug-resistant strains pose a significant public health threat due to their emergence and spread in clinical settings and the environment. The aim of this study was to determine a novel resistance mechanism against aminoglycoside antimicrobials in a clinical isolate.
METHODS
The function of was verified by gene cloning and antibiotic susceptibility tests. To explore the activity of the enzyme, recombinant proteins were expressed, and enzyme kinetics were tested. To determine the molecular background and mechanism of , whole-genome sequencing and bioinformatic analysis were performed.
RESULTS
The novel aminoglycoside -acetyltransferase gene confers resistance to several aminoglycosides. Among the antimicrobials tested, ribostamycin showed the highest increase (128-fold) in the minimum inhibitory concentration (MIC) compared with the control strains. According to the MIC results of the cloned , AAC(6')-Va also showed the highest catalytic efficiency for ribostamycin [/ ratio = (3.35 ± 0.17) × 10 M s]. Sharing the highest amino acid identity of 54.68% with AAC(6')-VaIc, the novel aminoglycoside -acetyltransferase constituted a new branch of the AAC(6') family due to its different resistance profiles. The gene context of and its close relatives was conserved in the genomes of species of the genus .
CONCLUSION
The novel resistance gene confers resistance to several aminoglycosides, especially ribostamycin. Our finding of a novel resistance gene in clinical will help us develop more effective treatments for this pathogen's infections.
PubMed: 37920263
DOI: 10.3389/fmicb.2023.1229593 -
Frontiers in Veterinary Science 2023Ramie (, BN) is used as livestock forage through suitable silage fermentation owing to its nutritional value. To date, relatively few studies have investigated the...
Ramie (, BN) is used as livestock forage through suitable silage fermentation owing to its nutritional value. To date, relatively few studies have investigated the effects of dietary fermented BN (FBN) on gut health in finishing pigs. The aim of the present study was to investigate the effects of dietary supplementation with 20% FBN on intestinal morphology, gene expression, and the functional response of the gut microbiota in finishing pigs. We found that FBN did not significantly affect serum antioxidant enzyme activities, ileal morphology, or the expression of genes encoding antioxidant enzymes, inflammatory cytokines, or tight junction proteins in the liver of the pigs. However, the gene expression levels of aryl hydrocarbon receptor () and interleukin 6 () were significantly downregulated in the ileum. A metagenomic analysis demonstrated that, compared with that seen in the control group, the cecal microbiota of pigs in the FBN treatment group was more closely clustered and contained a greater number of unique microbes. Bacteria were the predominant kingdom in the cecal microbiota, while Firmicutes, Bacteroidetes, and Proteobacteria were the dominant phyla, and , , and were the dominant genera. Dietary FBN significantly increased the abundance of the probiotic bacterium ( < 0.05). Functional analysis of the cecal microbiota showed that ABC transporter levels and glycolysis/gluconeogenesis-associated functions were diminished in FBN-fed pigs. Meanwhile, CAZyme analysis revealed that dietary FBN significantly downregulated the contents of carbohydrate-active enzymes, such as GT2, GH1, GH25, and GH13_31. In addition, cytochrome P450 analysis revealed that the abundance of CYP51 and CYP512 decreased with FBN treatment. An assessment of antibiotic resistance based on the Comprehensive Antibiotic Resistance Database (CARD) annotation indicated that the cecal microbes from pigs in the FBN treatment group had increased resistance to lincosamide, streptogramin, and chloramphenicol and reduced resistance to amikacin, isepamicin, neomycin, lividomycin, gentamicin, paromomycin, ribostamycin, and butirosin. Finally, virulence factor-related analysis showed that putative hemolysin-associated functions were decreased, whereas fibronectin-binding protein, flagella, and alginate-associated functions were increased. Taken together, our data showed that FBN supplementation exerted only minor effects on intestinal morphology and microbial community composition, suggesting that it is potentially safe for use as a supplement in the diets of finishing pigs. However, more studies are needed to validate its functionality.
PubMed: 37841475
DOI: 10.3389/fvets.2023.1253778 -
Frontiers in Microbiology 2023Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for...
BACKGROUND
Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention.
METHODS
The bacterium DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the gene and its phylogenetic relationship with other AMEs.
RESULTS
A novel aminoglycoside 3'--phosphotransferase gene designated was identified in DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3'--phosphotransferase APH(3')-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-/ DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3')-Id showed the highest catalytic efficiency for ribostamycin [ of (4.96 ± 1.63) × 10 M/s], followed by paromomycin [ of (2.18 ± 0.21) × 10 M/s], neomycin [ of (1.73 ± 0.20) × 10 M/s], and kanamycin [ of (1.10 ± 0.18) × 10 M/s]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3')-Id. No mobile genetic element (MGE) was discovered surrounding the gene.
CONCLUSION
In this work, a novel aminoglycoside 3'--phosphotransferase gene designated encoded in the chromosome of the environmental isolate DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.
PubMed: 37700861
DOI: 10.3389/fmicb.2023.1224464 -
Angewandte Chemie (International Ed. in... Jun 2023The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of...
The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of F NMR methods to characterize complex exchange processes with multiple excited states.
Topics: Neomycin; Riboswitch; Ligands; Anti-Bacterial Agents; Aminoglycosides
PubMed: 36970768
DOI: 10.1002/anie.202218064 -
ChemPlusChem Nov 2022High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC -modifications of a neomycin-sensing riboswitch...
High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.
Topics: Riboswitch; Neomycin; Ribostamycin; RNA; Paromomycin; Framycetin; Aminoglycosides; Anti-Bacterial Agents; Ligands; Oligonucleotides; Mass Spectrometry
PubMed: 36220343
DOI: 10.1002/cplu.202200256 -
Nature Synthesis Jul 2022Aminoglycosides (AGs) represent a large group of pseudoglycoside natural products, in which several different sugar moieties are harnessed to an aminocyclitol core. AGs...
Aminoglycosides (AGs) represent a large group of pseudoglycoside natural products, in which several different sugar moieties are harnessed to an aminocyclitol core. AGs constitute a major class of antibiotics that target the prokaryotic ribosome of many problematic pathogens. Hundreds of AGs have been isolated to date, with 1,3-diaminocyclohexanetriol, known as 2-deoxystreptamine (2-DOS), being the most abundant aglycon core. However, owning to their diverse and complex architecture, all AG-based drugs are either natural substances or analogues prepared by late-stage modifications. Synthetic approaches to AGs are rare and lengthy; most studies involve semi-synthetic reunion of modified fragments. Here we report a bottom-up chemical synthesis of the 2-DOS-based AG antibiotic ribostamycin, which proceeds in ten linear operations from benzene. A key enabling transformation involves a Cu-catalyzed, enantioselective, dearomative hydroamination, which set the stage for the rapid and selective introduction of the remaining 2-DOS heteroatom functionality. This work demonstrates how the combination of a tailored, dearomative logic and strategic use of subsequent olefin functionalizations can provide practical and concise access to the AG class of compounds.
PubMed: 36213185
DOI: 10.1038/s44160-022-00080-x -
ChemMedChem Jul 2022Modification at the 5''-position of 4,5-disubstituted aminoglycoside antibiotics (AGAs) to circumvent inactivation by aminoglycoside modifying enzymes (AMEs) is well...
Modification at the 5''-position of 4,5-disubstituted aminoglycoside antibiotics (AGAs) to circumvent inactivation by aminoglycoside modifying enzymes (AMEs) is well known. Such modifications, however, unpredictably impact activity and affect target selectivity thereby hindering drug development. A survey of 5''-modifications of the 4,5-AGAs and the related 5-O-furanosyl apramycin derivatives is presented. In the neomycin and the apralog series, all modifications were well-tolerated, but other 4,5-AGAs require a hydrogen bonding group at the 5''-position for maintenance of antibacterial activity. The 5''-amino modification resulted in parent-like activity, but reduced selectivity against the human cytosolic decoding A site rendering this modification unfavorable in paromomycin, propylamycin, and ribostamycin. Installation of a 5''-formamido group and, to a lesser degree, a 5''-ureido group resulted in parent-like activity without loss of selectivity. These lessons will aid the design of next-generation AGAs capable of circumventing AME action while maintaining high antibacterial activity and target selectivity.
Topics: Aminoglycosides; Anti-Bacterial Agents; Humans; Neomycin; Protein Synthesis Inhibitors; Ribosomes; Structure-Activity Relationship
PubMed: 35385605
DOI: 10.1002/cmdc.202200120 -
Frontiers in Microbiology 2021Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we...
Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we characterized a novel resistance gene named from an isolate of a novel species, R33 (CCTCC AB 2021339). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 2'--acetyltransferase. Whole-genome sequencing and comparative genomic analysis were performed to elucidate the molecular characteristics of the genome and the genetic context of the resistance gene-related sequences. Among the functionally characterized resistance genes, AAC(2')-If shares the highest amino acid sequence identity of 70.79% with AAC(2')-Ia. AAC(2')-If confers resistance to several aminoglycoside antibiotics, showing the highest resistance activity against ribostamycin and neomycin. The recombinant strain harboring (pUCP20-/DH5α) showed 256- and 128-fold increases in the minimum inhibitory concentration (MIC) levels to ribostamycin and neomycin, respectively, compared with those of the control strains (DH5α and pUCP20/DH5α). The results of the kinetic analysis of AAC(2')-If were consistent with the MIC results of the cloned with the highest catalytic efficiency for ribostamycin ( ratio = [3.72 ± 0.52] × 10 M s). Whole-genome sequencing demonstrated that the gene was located on the chromosome with a relatively unique genetic environment. Identification of a novel aminoglycoside resistance gene in a strain of a novel species will help us find ways to elucidate the complexity of resistance mechanisms in the microbial population.
PubMed: 34867838
DOI: 10.3389/fmicb.2021.711037 -
Frontiers in Microbiology 2020The emergence of infections caused by bacterial pathogens that are resistant to current antibiotic therapy is a critical healthcare challenge. Aminoglycosides are...
Exploration of Antibiotic Activity of Aminoglycosides, in Particular Ribostamycin Alone and in Combination With Ethylenediaminetetraacetic Acid Against Pathogenic Bacteria.
The emergence of infections caused by bacterial pathogens that are resistant to current antibiotic therapy is a critical healthcare challenge. Aminoglycosides are natural antibiotics with broad spectrum of activity; however, their clinical use is limited due to considerable nephrotoxicity. Moreover, drug-resistant bacteria that cause infections in human as well as livestock are less responsive to conventional antibiotics. Herein, we report the antibacterial evaluation of five different aminoglycosides, including ribostamycin, against a panel of Gram-positive and Gram-negative pathogens. Eight of the tested bacterial strains are linked to gastrointestinal (GI) infections. The minimum inhibitory concentration (MIC) of ribostamycin against three different strains is in the range of 0.9-7.2 μM and against a strain of is 0.5 μM. We also found that the MIC of ribostamycin was considerably enhanced from 57.2 to 7.2 μM, an 8-fold improvement, when bacteria were treated with a combination of ribostamycin and ethylenediaminetetraacetic acid (EDTA). These findings demonstrate a promising approach to enhance the clinical potential of ribostamycin and provide a rational for its antibiotic reclassification from special level to non-restricted level.
PubMed: 32849365
DOI: 10.3389/fmicb.2020.01718