Epidemiologic, Phenotypic, and Structural Characterization of Aminoglycoside-Resistance Gene aac(3)-IV

Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure-activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections

In vitro susceptibility of Neisseria gonorrhoeae to netilmicin and etimicin in comparison to gentamicin and other aminoglycosides

PURPOSE Single doses of gentamicin have demonstrated clinical efficacy in the treatment of urogenital gonorrhea, but lower cure rates for oropharyngeal and anorectal gonorrhea. Formulations selectively enriched in specific gentamicin C congeners have been proposed as a less toxic alternative to gentamicin, potentially permitting higher dosing to result in increased plasma exposures at the extragenital sites of infection. The purpose of the present study was to compare the antibacterial activity of individual gentamicin C congeners against Neisseria gonorrhoeae to that of other aminoglycoside antibiotics. METHODS Antimicrobial susceptibility of three N. gonorrhoeae reference strains and 152 clinical isolates was assessed using standard disk diffusion, agar dilution, and epsilometer tests. RESULTS Gentamicin C1, C2, C1a, and C2a demonstrated similar activity against N. gonorrhoeae. Interestingly, susceptibility to the 1-N-ethylated aminoglycosides etimicin and netilmicin was significantly higher than the susceptibility to their parent compounds gentamicin C1a and sisomicin, and to any other of the 25 aminoglycosides assessed in this study. Propylamycin, a 4'-propylated paromomycin analogue, was significantly more active against N. gonorrhoeae than its parent compound, too. CONCLUSION Selectively enriched gentamicin formulations hold promise for a less toxic but equally efficacious alternative to gentamicin. Our study warrants additional consideration of the clinically established netilmicin and etimicin for treatment of genital and perhaps extragenital gonorrhea. Additional studies are required to elucidate the mechanism behind the advantage of alkylated aminoglycosides

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates among individuals at high risk for sexually transmitted diseases in Zurich, Switzerland

Background: While ceftriaxone resistance remains scarce in Switzerland, global Neisseria gonorrhoeae (NG) antimicrobial resistance poses an urgent threat. This study describes clinical characteristics in MSM (men who have sex with men) diagnosed with NG infection and analyses NG resistance by phenotypic and genotypic means. Methods: Data of MSM enrolled in three clinical cohorts with a positive polymerase chain reaction test (PCR) for NG were analysed between January 2019 and December 2021 and linked with antibiotic susceptibility testing. Bacterial isolates were subjected to whole genome sequencing (WGS). Results: Of 142 participants, 141 (99%) were MSM and 118 (84%) living with HIV. Participants were treated with ceftriaxone ( N = 79), azithromycin ( N = 2), or a combination of both ( N = 61). No clinical or microbiological failures were observed. From 182 positive PCR samples taken, 23 were available for detailed analysis. Based on minimal inhibitory concentrations (MICs), all isolates were susceptible to ceftriaxone, gentamicin, cefixime, cefpodoxime, ertapenem, zoliflodacin, and spectinomycin. Resistance to azithromycin, tetracyclines and ciprofloxacin was observed in 10 (43%), 23 (100%) and 11 (48%) of the cases, respectively. Analysis of WGS data revealed combinations of resistance determinants that matched with the corresponding phenotypic resistance pattern of each isolate. Conclusion: Among the MSM diagnosed with NG mainly acquired in Switzerland, ceftriaxone MICs were low for a subset of bacterial isolates studied and no treatment failures were observed. For azithromycin, high occurrences of in vitro resistance were found. Gentamicin, cefixime, cefpodoxime, ertapenem, spectinomycin, and zoliflodacin displayed excellent in vitro activity against the 23 isolates underscoring their potential as alternative agents to ceftriaxone

Apramycin susceptibility of multidrug-resistant Gram-negative blood culture isolates in five countries in South-East Asia

Bloodstream infections (BSIs) are a leading cause of sepsis, a life-threatening condition that contributes significantly to the mortality of bacterial infections. Aminoglycoside antibiotics such as gentamicin or amikacin are essential medicines in the treatment of BSIs, but their clinical efficacy is increasingly compromised by antimicrobial resistance. The aminoglycoside apramycin has demonstrated preclinical efficacy against aminoglycoside- and multidrug-resistant (MDR) Gram-negative bacilli (GNB) and is currently in clinical development for the treatment of critical systemic infections. Here, we collected a panel of 470 MDR GNB isolates from health care facilities in Cambodia, Laos, Singapore, Thailand, and Vietnam for a multi-centre assessment of their antimicrobial susceptibility to apramycin in comparison to other aminoglycosides and colistin by broth microdilution assays. Apramycin and amikacin MICs ≤ 16 µg/mL were found for 462 (98.3%) and 408 (86.8%) GNB isolates, respectively. Susceptibility to gentamicin and tobramycin (MIC ≤ 4 µg/mL) was significantly lower at 122 (26.0%) and 101 (21.5%) susceptible isolates, respectively. Of note, all carbapenem- and third-generation cephalosporin (3GC) resistant Enterobacterales, all Acinetobacter baumannii, and all Pseudomonas aeruginosa isolates tested in this study appeared to be susceptible to apramycin. Of the 65 colistin-resistant isolates tested, only four (6.2%) had an apramycin MIC > 16 µg/mL. Apramycin demonstrated best-in-class activity against a panel of GNB isolates with resistances to other aminoglycosides, carbapenems, 3GC, and colistin, warranting continued consideration of apramycin as a drug candidate for the treatment of multidrug-resistant BSIs. Keywords: Bloodstream infection; Gram negative; aminoglycoside; antimicrobial resistance; apramycin; blood culture isolates

Delivering Sustained, Coordinated, and Integrated Observations of the Southern Ocean for Global Impact

The Southern Ocean is disproportionately important in its effect on the Earth system, impacting climatic, biogeochemical and ecological systems, which makes recent observed changes to this system cause for global concern. The enhanced understanding and improvements in predictive skill needed for understanding and projecting future states of the Southern Ocean require sustained observations. Over the last decade, the Southern Ocean Observing System (SOOS) has established networks for enhancing regional coordination and research community groups to advance development of observing system capabilities. These networks support delivery of the SOOS 20-year vision, which is to develop a circumpolar system that ensures time series of key variables, and deliver the greatest impact from data to all key end-users. Although the Southern Ocean remains one of the least-observed ocean regions, enhanced international coordination and advances in autonomous platforms have resulted in progress towards addressing the need for sustained observations of this region. Since 2009, the Southern Ocean community has deployed over 5700 observational platforms south of 40°S. Large-scale, multi-year or sustained, multidisciplinary efforts have been supported and are now delivering observations of essential variables at space and time scales that enable assessment of changes being observed in Southern Ocean systems. The improved observational coverage, however, is predominantly for the open ocean, encompasses the summer, consists of primarily physical oceanographic variables and covers surface to 2000 m. Significant gaps remain in observations of the ice-impacted ocean, the sea ice, depths more than 2000 m, the air-sea-ice interface, biogeochemical and biological variables, and for seasons other than summer. Addressing these data gaps in a sustained way requires parallel advances in coordination networks, cyberinfrastructure and data management tools, observational platform and sensor technology, platform interrogation and data-transmission technologies, modeling frameworks, and internationally agreed sampling requirements of key variables. This paper presents a community statement on the major scientific and observational progress of the last decade, and importantly, an assessment of key priorities for the coming decade, towards achieving the SOOS vision and delivering essential data to all end users

Epidemiologic, Phenotypic, and Structural Characterization of Aminoglycoside-Resistance Gene aac(3)-IV

Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure–activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections

Optimization of the antimicrobial peptide Bac7 by deep mutational scanning

Background: Intracellularly active antimicrobial peptides are promising candidates for the development of antibiotics for human applications. However, drug development using peptides is challenging as, owing to their large size, an enormous sequence space is spanned. We built a high-throughput platform that incorporates rapid investigation of the sequence-activity relationship of peptides and enables rational optimization of their antimicrobial activity. The platform is based on deep mutational scanning of DNA-encoded peptides and employs highly parallelized bacterial self-screening coupled to next-generation sequencing as a readout for their antimicrobial activity. As a target, we used Bac71-23, a 23 amino acid residues long variant of bactenecin-7, a potent translational inhibitor and one of the best researched proline-rich antimicrobial peptides. Results: Using the platform, we simultaneously determined the antimicrobial activity of >600,000 Bac71-23 variants and explored their sequence-activity relationship. This dataset guided the design of a focused library of ~160,000 variants and the identification of a lead candidate Bac7PS. Bac7PS showed high activity against multidrug-resistant clinical isolates of E. coli, and its activity was less dependent on SbmA, a transporter commonly used by proline-rich antimicrobial peptides to reach the cytosol and then inhibit translation. Furthermore, Bac7PS displayed strong ribosomal inhibition and low toxicity against eukaryotic cells and demonstrated good efficacy in a murine septicemia model induced by E. coli. Conclusion: We demonstrated that the presented platform can be used to establish the sequence-activity relationship of antimicrobial peptides, and showed its usefulness for hit-to-lead identification and optimization of antimicrobial drug candidates. Keywords: Antibiotics; Antimicrobial peptides; Antimicrobial resistance; Antimicrobials; Deep mutational scanning; Drug discovery; High-throughput screening; Proline-rich antimicrobial peptides; Protein synthesis inhibitor; Sequence-activity relationship

Antimicrobial susceptibility patterns of respiratory Gram-negative bacterial isolates from COVID-19 patients in Switzerland

BACKGROUND Bacterial superinfections associated with COVID-19 are common in ventilated ICU patients and impact morbidity and lethality. However, the contribution of antimicrobial resistance to the manifestation of bacterial infections in these patients has yet to be elucidated. METHODS We collected 70 Gram-negative bacterial strains, isolated from the lower respiratory tract of ventilated COVID-19 patients in Zurich, Switzerland between March and May 2020. Species identification was performed using MALDI-TOF; antibiotic susceptibility profiles were determined by EUCAST disk diffusion and CLSI broth microdilution assays. Selected Pseudomonas aeruginosa isolates were analyzed by whole-genome sequencing. RESULTS Pseudomonas aeruginosa (46%) and Enterobacterales (36%) comprised the two largest etiologic groups. Drug resistance in P. aeruginosa isolates was high for piperacillin/tazobactam (65.6%), cefepime (56.3%), ceftazidime (46.9%) and meropenem (50.0%). Enterobacterales isolates showed slightly lower levels of resistance to piperacillin/tazobactam (32%), ceftriaxone (32%), and ceftazidime (36%). All P. aeruginosa isolates and 96% of Enterobacterales isolates were susceptible to aminoglycosides, with apramycin found to provide best-in-class coverage. Genotypic analysis of consecutive P. aeruginosa isolates in one patient revealed a frameshift mutation in the transcriptional regulator nalC that coincided with a phenotypic shift in susceptibility to β-lactams and quinolones. CONCLUSIONS Considerable levels of antimicrobial resistance may have contributed to the manifestation of bacterial superinfections in ventilated COVID-19 patients, and may in some cases mandate consecutive adaptation of antibiotic therapy. High susceptibility to amikacin and apramycin suggests that aminoglycosides may remain an effective second-line treatment of ventilator-associated bacterial pneumonia, provided efficacious drug exposure in lungs can be achieved