APH(3')-Ie, an aminoglycoside-modifying enzyme discovered in a rabbit-derived Citrobacter gillenii isolate.

Background: Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms. Methods: The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques. Results: A novel aminoglycoside resistance gene, designated aph(3')-Ie, which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (K m, 4.22 ± 0.88 µM) and the highest catalytic efficiency [k cat/K m, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3') proteins, APH(3')-Ie contained all the conserved functional sites of the APH family. The aph(3')-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance. Conclusion: In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3')-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.

Prevalence of resistance to macrolides and aminoglycosides in Mycobacterium avium, M. abscessus, and M. chelonae identified in the Laboratorio Nacional de Referencia of Colombia from 2018 to 2022 / Prevalencia de la resistencia a macrólidos y aminoglucósidos en los complejos Mycobacterium avium y M. abscessus y en Mycobacterium chelonae identificados en el Laboratorio Nacional de Referencia de Colombia entre el 2018 y el 2022.

Introduction: The Mycobacterium chelonae species and the M. avium and M. abscessus complexes are emerging pathogens that cause mycobacteriosis. Treatment depends on the species and subspecies identified. The drugs of choice are macrolides and aminoglycosides. However, due to the resistance identified to these drugs, determining the microbe's sensitivity profile will allow clinicians to improve the understanding of the prognosis and evolution of these pathologies. Objective: To describe the macrolide and aminoglycoside susceptibility profile of cultures identified by Colombia's Laboratorio Nacional de Referencia de Mycobacteria from 2018 to 2022, as Mycobacterium avium complex, M. abscessus complex, and M. chelonae. Materials and methods. This descriptive study exposes the susceptibility profile to macrolides and aminoglycosides of cultures identified as M. avium complex, M. abscessus complex, and M. chelonae using the GenoType® NTM-DR method. Materials and methods: This descriptive study exposes the susceptibility profile to macrolides and aminoglycosides of cultures identified as M. avium complex, M. abscessus complex, and M. chelonae using the GenoType® NTM-DR method. Results: We identified 159 (47.3 %) cultures as M. avium complex, of which 154 (96.9 %) were sensitive to macrolides, and 5 (3.1 %) were resistant; all were sensitive to aminoglycosides. From the 125 (37.2 %) cultures identified as M. abscessus complex, 68 (54.4 %) were sensitive to macrolides, 57 (45.6 %) were resistant to aminoglycosides, and just one (0.8 %) showed resistance to aminoglycosides. The 52 cultures (15.5 %) identified as M. chelonae were sensitive to macrolides and aminoglycosides. Conclusions: The three studied species of mycobacteria have the least resistance to Amikacin. Subspecies identification and their susceptibility profiles allow the establishment of appropriate treatment schemes, especially against M. abscessus.

Introducción. Mycobacterium chelonae y los complejos Mycobacterium avium y M. abscessus, son agentes patógenos emergentes causantes de micobacteriosis. El tratamiento de esta infección depende de la especie y la subespecie identificadas. Los fármacos de elección son los macrólidos y aminoglucósidos, contra los cuales se ha reportado resistencia; por esta razón, el determinar el perfil de sensibilidad le permite al médico tratante comprender mejor el pronóstico y la evolución de estas infecciones. Objetivo. Describir los perfiles de sensibilidad ante macrólidos y aminoglucósidos, de los cultivos identificados como complejo Mycobacterium avium, complejo M. abscessus o especie M. chelonae, en el Laboratorio Nacional de Referencia de Micobacterias durante los años 2018 a 2022. Materiales y métodos. Se llevó a cabo un estudio descriptivo del perfil de sensibilidad a macrólidos y aminoglucósidos, de los cultivos identificados como complejo M. avium, complejo M. abscessus o M. chelonae, mediante la metodología GenoType® NTM-DR. Resultados. Los cultivos del complejo M. avium fueron 159 (47,3 %), de los cuales, 154 (96,9 %) fueron sensibles y 5 (3,1 %) resistentes a los macrólidos; todos fueron sensibles a los aminoglucósidos. Del complejo M. abscessus se estudiaron 125 (37,2 %) cultivos, 68 (54,4 %) resultaron sensibles y 57 (45,6 %) resistentes a los macrólidos; solo un cultivo (0,8 %) fue resistente a los aminoglucósidos. De M. chelonae se analizaron 52 cultivos (15,5 %), todos sensibles a los macrólidos y aminoglucósidos. Conclusiones. En las tres especies de micobacterias estudiadas, la resistencia contra la amikacina fue la menos frecuente. La identificación de las subespecies y los perfiles de sensibilidad permiten instaurar esquemas de tratamiento adecuados, especialmente en las micobacteriosis causadas por M. abscessus.

[Assessment of the risk of hearing loss in children with cystic fibrosis]. / Otsenka riska patologii slukha pri mukovistsidoze u detei.

BACKGROUND: Cystic fibrosis (CF) is a severe hereditary disease with a multisystem lesion. Manifestations of CF include severe infectious purulent lesions of all parts of the respiratory tract, including purulent rhinosinusitis with nasal polyps. The involvement of the sinonasal region and the need for systemic use of ototoxic drugs (primarily aminoglycosides to treat resistant bacterial infection) potentially create a risk of both conductive and sensorineural hearing loss (SNHL). The available data on the epidemiology of hearing disorders in CF is contradictory. Currently, genetic determinants of the development of aminoglycoside SNHL have been identified. MATERIAL AND METHODS: For 136 CF patients (75 girls, 61 boys) aged 3 to 17 (9.4±3.9) years were performed audiological examination: tympanometry, transient-evoked otoacoustic emission and the pure tone threshold audiometry (standard frequency range) (n=126). History of systemic therapy with aminoglycosides was evaluated for each patient. Sequencing of c.35delG mutations in the GJB2 gene (nuclear DNA) and A1555G in the 12S rRNA gene (mitochondrial DNA) was performed in 215 patients with cystic fibrosis (the group partially overlaps with the audiological group), and as a control - 106 children with bronchial asthma and 103 healthy children, their age ranged from 3 to 17 (8.8±3.8) years. RESULTS: Audiological examination of CF children reveled a prevalence of conductive hearing loss comparable to the general population (2.4%). The frequency of SNHL was 1.6%, wich exceeds that of non-CF children. A genetic study revealed one case of heterozygous carriage of the c.35delG mutation in the GJB2 gene in a patient with bronchial asthma. In the group of patients with CF (n=215), mutations in the connexin 26 gene were not detected. No A1555G mutation was detected either in the group of patients with CF or in the control groups. CONCLUSIONS: Children with CF are at risk for the development of sensorineural, but not conductive hearing loss. Routine total screening for A1555G and c.35delG mutations probably seems not to be recommended.

Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance.

Aminoglycoside antibiotics target ribosomes and are effective against a wide range of bacteria. Here, we demonstrated that knockout strains related to energy metabolism in Escherichia coli showed increased tolerance to aminoglycosides during the mid-exponential growth phase. Contrary to expectations, these mutations did not reduce the proton motive force or aminoglycoside uptake, as there were no significant changes in metabolic indicators or intracellular gentamicin levels between wild-type and mutant strains. Our comprehensive proteomics analysis unveiled a noteworthy upregulation of proteins linked to the tricarboxylic acid (TCA) cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research provides valuable insights into the mechanisms of aminoglycoside tolerance, paving the way for novel strategies to combat such cells.

Bacteria that are resistant to antibiotic drugs pose a significant challenge to human health around the globe. They have acquired genetic mutations that allow them to survive and grow in the presence of one or more antibiotics, making it harder for clinicians to eliminate such bacteria from human patients with life-threatening infections. Some bacteria may be able to temporarily develop tolerance to an antibiotic by altering how they grow and behave, without acquiring any new genetic mutations. Such drug-tolerant bacteria are more likely to survive long enough to gain mutations that may promote drug resistance. Recent studies suggest that genes involved in processes collectively known as energy metabolism, which convert food sources into the chemical energy cells need to survive and grow, may play a role in both tolerance and resistance. For example, Escherichia coli bacteria develop mutations in energy metabolism genes when exposed to members of a family of antibiotics known as the aminoglycosides. However, it remains unclear what exact role energy metabolism plays in antibiotic tolerance. To address this question, Shiraliyev and Orman studied how a range of E. coli strains with different genetic mutations affecting energy metabolism could survive in the presence of aminoglycosides. The experiments found that most of the mutant strains had a higher tolerance to the drugs than normal E. coli. Unexpectedly, this increased tolerance did not appear to be due to the drugs entering the mutant bacterium cells less than they enter normal cells (a common strategy of drug resistance and tolerance). Further experiments using a technique, known as proteomics, revealed that many genes involved in energy metabolism were upregulated in the mutant bacteria, suggesting these cells were compensating for the genetic abnormalities they have. Furthermore, the mutant bacteria had lower levels of the molecules the antibiotics target than normal bacteria. The findings of Shiraliyev and Orman offer critical insights into how bacteria become tolerant of aminoglycoside antibiotics. In the future, this may guide the development of new strategies to combat bacterial diseases.

Piplartine attenuates aminoglycoside-induced TRPV1 activity and protects from hearing loss in mice.

Hearing loss is a major health concern in our society, affecting more than 400 million people worldwide. Among the causes, aminoglycoside therapy can result in permanent hearing loss in 40% to 60% of patients receiving treatment, and despite these high numbers, no drug for preventing or treating this type of hearing loss has yet been approved by the US Food and Drug Administration. We have previously conducted high-throughput screenings of bioactive compounds, using zebrafish as our discovery platform, and identified piplartine as a potential therapeutic molecule. In the present study, we expanded this work and characterized piplartine's physicochemical and therapeutic properties. We showed that piplartine had a wide therapeutic window and neither induced nephrotoxicity in vivo in zebrafish nor interfered with aminoglycoside antibacterial activity. In addition, a fluorescence-based assay demonstrated that piplartine did not inhibit cytochrome C activity in microsomes. Coadministration of piplartine protected from kanamycin-induced hair cell loss in zebrafish and protected hearing function, outer hair cells, and presynaptic ribbons in a mouse model of kanamycin ototoxicity. Last, we investigated piplartine's mechanism of action by phospho-omics, immunoblotting, immunohistochemistry, and molecular dynamics experiments. We found an up-regulation of AKT1 signaling in the cochleas of mice cotreated with piplartine. Piplartine treatment normalized kanamycin-induced up-regulation of TRPV1 expression and modulated the gating properties of this receptor. Because aminoglycoside entrance to the inner ear is, in part, mediated by TRPV1, these results suggested that by regulating TRPV1 expression, piplartine blocked aminoglycoside's entrance, thereby preventing the long-term deleterious effects of aminoglycoside accumulation in the inner ear compartment.

Are Aminoglycoside Antibiotics TRPing Your Metabolic Switches?

Transient receptor potential (TRP) channels are broadly implicated in the developmental programs of most tissues. Amongst these tissues, skeletal muscle and adipose are noteworthy for being essential in establishing systemic metabolic balance. TRP channels respond to environmental stimuli by supplying intracellular calcium that instigates enzymatic cascades of developmental consequence and often impinge on mitochondrial function and biogenesis. Critically, aminoglycoside antibiotics (AGAs) have been shown to block the capacity of TRP channels to conduct calcium entry into the cell in response to a wide range of developmental stimuli of a biophysical nature, including mechanical, electromagnetic, thermal, and chemical. Paradoxically, in vitro paradigms commonly used to understand organismal muscle and adipose development may have been led astray by the conventional use of streptomycin, an AGA, to help prevent bacterial contamination. Accordingly, streptomycin has been shown to disrupt both in vitro and in vivo myogenesis, as well as the phenotypic switch of white adipose into beige thermogenic status. In vivo, streptomycin has been shown to disrupt TRP-mediated calcium-dependent exercise adaptations of importance to systemic metabolism. Alternatively, streptomycin has also been used to curb detrimental levels of calcium leakage into dystrophic skeletal muscle through aberrantly gated TRPC1 channels that have been shown to be involved in the etiology of X-linked muscular dystrophies. TRP channels susceptible to AGA antagonism are critically involved in modulating the development of muscle and adipose tissues that, if administered to behaving animals, may translate to systemwide metabolic disruption. Regenerative medicine and clinical communities need to be made aware of this caveat of AGA usage and seek viable alternatives, to prevent contamination or infection in in vitro and in vivo paradigms, respectively.

Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated. MATERIALS AND METHODS: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates. RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13). CONCLUSIONS: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

A computational workflow to determine drug candidates alternative to aminoglycosides targeting the decoding center of E. coli ribosome.

The global antibiotic resistance problem necessitates fast and effective approaches to finding novel inhibitors to treat bacterial infections. In this study, we propose a computational workflow to identify plausible high-affinity compounds from FDA-approved, investigational, and experimental libraries for the decoding center on the small subunit 30S of the E. coli ribosome. The workflow basically consists of two molecular docking calculations on the intact 30S, followed by molecular dynamics (MD) simulations coupled with MM-GBSA calculations on a truncated ribosome structure. The parameters used in the molecular docking suits, Glide and AutoDock Vina, as well as in the MD simulations with Desmond were carefully adjusted to obtain expected interactions for the ligand-rRNA complexes. A filtering procedure was followed, considering a fingerprint based on aminoglycoside's binding site on the 30S to obtain seven hit compounds either with different clinical usages or aminoglycoside derivatives under investigation, suggested for in vitro studies. The detailed workflow developed in this study promises an effective and fast approach for the estimation of binding free energies of large protein-RNA and ligand complexes.

Quantitative analysis of the impurities in Etimicin using hydrophilic interaction liquid chromatography coupled with charged aerosol detector.

Etimicin is a typical aminoglycoside antibiotic (AG). High performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD) method is a commonly used method for determining impurities in Etimicin. However, due to the poor reproducibility, low sensitivity and narrow linear range of the ELSD, high-throughput quantitative analysis of impurities in Etimicin currently poses a challenge. In this study, a sensitive method using hydrophilic interaction liquid chromatography coupled with charged aerosol detector (HILIC-CAD) was developed for the analysis of the impurities in Etimicin. The liquid phase conditions for determination impurities in Etimicin were optimized using Box Behnken design (BBD) and response surface methodology (RSM), resulting in satisfactory separation and optimal CAD output signal. We also studied the influence of CAD parameters on the signal-to-noise ratio and linearity of Etimicin and its impurities. This method has also been proven to be effective in separating impurities from two other typical AGs, Isepamicin and Amikacin. In the method validation, the coefficient of determination (R2) of Etimicin, Isepamicin and Amikacin and their impurities were all greater than 0.999, within the range of 0.5-50 µg/mL. The average recoveries of the impurities of three typical AGs were 99.03 %-101.22 %, RSDs all were less than 2.5 % for intra-day and inter-day precision, with good precision and accuracy. The developed HILIC-CAD quantification method was sensitive, accurate and highly selective for quantitative analysis of impurities in the AGs without need ion-pairing reagents, which is ensure the public medication safety. The method is first reported application of HILIC-CAD method for quantitative analysis of the impurities in AGs.

Sophisticated natural products as antibiotics.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

PBI derivatives/surfactant-based fluorescent ensembles: Sensing of multiple aminoglycoside antibiotics and interaction mechanism studies.

Fluorescent aggregates and ensembles have been widely applied in fabrication of fluorescent sensors due to their capacity of encapsulating fluorophores and modulating their photophysical properties. In the present work, fluorescent ensembles based on anionic surfactant SDS assemblies and perylene derivatives (PBIs) were particularly constructed. Three newly synthesized neutral PBI derivatives with different structures, PO, PC1 and PC2, were used for the purpose to evaluate probe structure influence on constructing fluorescent ensembles. The one with hydrophilic side chains, PO, experienced distinct photophysical modulation effect by SDS assemblies. The ensemble based on PO@SDS assemblies displayed effective fluorescence variation to antibiotic aminoglycosides (AGs). To improve cross-reactivity and discrimination capability of ensembles, a second probe, coumarin, was introduced into PO@SDS assemblies. The resultant ternary sensor, CM-PO@SDS, exhibited good qualitative and quantitative detection capabilities, and achieved differentiation of eight AGs and mixed AG samples both in aqueous solution and actual biological fluid, like human serum. Sensing mechanism studies revealed that hydrogen bonding, electrostatic and hydrophobic interactions are involved in the sensing process. This surfactant-based fluorescent ensemble provides a simple and feasible method for assessing AGs levels. Meanwhile, this work may provide some insights to design reasonable probes for constructing effective single-system based discriminative fluorescent amphiphilic sensors.

Phenotypic and genotypic assessment of fluoroquinolones and aminoglycosides resistances in Pseudomonas aeruginosa collected from Minia hospitals, Egypt during COVID-19 pandemic.

BACKGROUND: One of the most prevalent bacteria that cause nosocomial infections is Pseudomonas aeruginosa. Fluoroquinolones (FQ) and aminoglycosides are vital antipseudomonal drugs, but resistance is increasingly prevalent. The study sought to investigate the diverse mechanisms underlying FQ and aminoglycoside resistance in various P. aeruginosa strains particularly during the COVID-19 crisis. METHODS: From various clinical and environmental samples, 110 P. aeruginosa isolates were identified and their susceptibility to several antibiotic classes was evaluated. Molecular techniques were used to track target gene mutations, the presence of genes encoding for quinolone resistance, modifying enzymes for aminoglycosides and resistance methyltransferase (RMT). Efflux pump role was assessed phenotypically and genotypically. Random amplified polymorphic DNA (RAPD) analysis was used to measure clonal diversity. RESULTS: QnrS was the most frequently encountered quinolone resistance gene (37.5%) followed by qnrA (31.2%) and qnrD (25%). Among aminoglycoside resistant isolates, 94.1% harbored modifying enzymes genes, while RMT genes were found in 55.9% of isolates. The aac(6')-Ib and rmtB were the most prevalent genes (79.4% and 32.3%, respectively). Most FQ resistant isolates overexpressed mexA (87.5%). RAPD fingerprinting showed 63.2% polymorphism. CONCLUSIONS: Aminoglycosides and FQ resistance observed in this study was attributed to several mechanisms with the potential for cross-contamination existence so, strict infection control practices are crucial.

An open-label, randomized, non-inferiority trial of the efficacy and safety of ciprofloxacin versus an aminoglycoside + ciprofloxacin in the treatment of bubonic plague (IMASOY): study protocol for a randomized control trial-an update to the published protocol.

This article reports an update to the protocol of the IMASOY trial, which was prospectively registered on clinicaltrials.gov (NCT04110340) in October 2019.

Prevalence and mechanisms of aminoglycoside resistance among drug-resistant Pseudomonas aeruginosa clinical isolates in Iran.

BACKGROUND: Aminoglycosides have been a cornerstone of the treatment of nosocomial infections caused by Pseudomonas aeruginosa for over 80 years. However, escalating emergence of resistance poses a significant challenge. Therefore, this study aimed to investigate the prevailing patterns of aminoglycoside resistance among clinical isolates of P. aeruginosa in Iran; as well as the underlying resistance mechanisms observed in patients referred to Ardabil hospitals. METHODS: A total of 200 isolates from five hospitals were evaluated. The resistance profiles of P. aeruginosa isolates to tobramycin, amikacin, and netilmicin were determined using the disk diffusion method. The capacity of aminoglycoside-resistant isolates to form biofilms was assessed through a phenotypic assay, and the results were confirmed using the gene amplification technique. The presence of genes associated with aminoglycoside resistance was detected using polymerase chain reaction (PCR). Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression levels of genes encoding the MexXY-OprM efflux pump and PhoPQ two-component system (TCS). RESULTS: The prevalence of aminoglycoside-resistant P. aeruginosa isolates was 48%, with 94.7% demonstrating multidrug resistance (MDR). All aminoglycoside-resistant P. aeruginosa strains exhibited biofilm-forming capabilities and harbored all the genes associated with biofilm production. Among the nine genes encoding 16S rRNA methylase and aminoglycoside-modifying enzymes, three genes were detected in these isolates: aac(6')-Ib (85.4%), ant(2'')-Ia (18.7%), and aph(3')-VI (3.1%). Additionally, all aminoglycoside-resistant P. aeruginosa isolates carried mexY and phoP genes, although the expression levels of mexY and phoP were 75% and 87.5%, respectively. CONCLUSION: Given the considerably high prevalence of aminoglycoside-resistant P. aeruginosa strains, urgent measures are warranted to transition towards the use of novel aminoglycosides and to uphold vigilant surveillance of resistance patterns.

Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-ManpN3NA.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-ß-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The ß-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

Hyperosmotic sisomicin infusion: a mouse model for hearing loss.

Losing either type of cochlear sensory hair cells leads to hearing impairment. Inner hair cells act as primary mechanoelectrical transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established inner ear damage models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 h, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained until three days post-treatment, after which deterioration in structure and number was observed, culminating in a complete hair cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.

Determination of Aminoglycosides by Ion-Pair Liquid Chromatography with UV Detection: Application to Pharmaceutical Formulations and Human Serum Samples.

Aminoglycosides (AGs) represent a prominent class of antibiotics widely employed for the treatment of various bacterial infections. Their widespread use has led to the emergence of antibiotic-resistant strains of bacteria, highlighting the need for analytical methods that allow the simple and reliable determination of these drugs in pharmaceutical formulations and biological samples. In this study, a simple, robust and easy-to-use analytical method for the simultaneous determination of five common aminoglycosides was developed with the aim to be widely applicable in routine laboratories. With this purpose, different approaches based on liquid chromatography with direct UV spectrophotometric detection methods were investigated: on the one hand, the use of stationary phases based on hydrophilic interactions (HILIC); on the other hand, the use of reversed-phases in the presence of an ion-pairing reagent (IP-LC). The results obtained by HILIC did not allow for an effective separation of aminoglycosides suitable for subsequent spectrophotometric UV detection. However, the use of IP-LC with a C18 stationary phase and a mobile phase based on tetraborate buffer at pH 9.0 in the presence of octanesulfonate, as an ion-pair reagent, provided adequate separation for all five aminoglycosides while facilitating the use of UV spectrophotometric detection. The method thus developed, IP-LC-UV, was optimized and applied to the quality control of pharmaceutical formulations with two or more aminoglycosides. Furthermore, it is demonstrated here that this methodology is also suitable for more complex matrices, such as serum, which expands its field of application to therapeutic drug monitoring, which is crucial for aminoglycosides, with a therapeutic index ca. 50%.