Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis

COVID-19; SARS-CoV-2; Electronic health recordsCOVID-19; SARS-CoV-2; Registros médicos electrónicosCOVID 19; SARS-CoV-2; Registres mèdics electrònicsBackground Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance.This work was supported by MSD and by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU through grants PI21/00017 and Personalized and precision medicine grant (MePRAM Project, PMP22/00092)

In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

[eng] Pseudomonas aeruginosa, is a major cause of nosocomial and chronic infections, being considered a paradigm of antimicrobial resistance development. However, in the present study the dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in wild-type (PAO1) and mutator P. aeruginosa (PAOMS, ΔmutS) have been compared. The strains were incubated in Müeller-Hinton Broth (MHB) for 24h with 0.125 to 64 µg/mL of imipenem, and imipenem/relebactam at 4 µg/mL fixed concentration. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 µg/mL of imipenem for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed for 40 derived mutants from PAO1 and 40 from PAOMS. Those mutants were further characterized by whole-genome sequencing (WGS) through DNA isolation, and its virulence studied by Caenorhabditis elegans model. Development of high-level imipenem resistance development was faster for PAOMS than PAO1 mutants. Furthermore, when both strains were treated with imipenem/relebactam, development resistance to imipenem was reduced. Moreover, characterization of mutants by WGS indicated that most of mutants which were resistant to imipenem, had mutations on oprD and ampC, while imipenem/relebactam resistant mutants had mutations in oprD and MexAB-OprM efflux system genes. Additionally, virulence and lethality of PAO1 and PAOMS mutants tested by C. elegans model demonstrated that those more resistant mutants were non-virulent, and the more sensitive ones were considered virulent. Those results suggested relebactam as an adequate partner for imipenem to remedy infections caused by P. aeruginosa resistant to carbapenems.[spa] P. aeruginosa es una de las mayores causas de infecciones nosocomiales y crónicas, siendo un paradigma del desarrollo de resistencias a antibióticos. Así pues, en el presente estudio se compara la dinámica y el desarrollo de mecanismos de resistencia a imipenem e imipenem/relebactam en cepas de P. aeruginosa PAO1, así como de P. aeruginosaΔmutS conocida como PAOMS. Ambas cepas fueron incubadas en caldo Müeller-Hinton por separado durante 24h, a una concentración creciente de imipenem de 0.125 a 64 µg/mL, e imipenem/relebactam a concentración constante de este último de 4 µg/mL. Los tubos de mayor concentración de antibiótico con crecimiento visible fueron reinoculados en medio fresco con concentraciones superiores de imipenem, repitiéndose el mismo procedimiento durante 7 días consecutivos. Los perfiles de sensibilidad y mecanismos de resistencia se estudiaron de cada uno de los mutantes obtenidos, siendo caracterizados por secuenciación del genoma entero a partir del aislamiento de ADN. Además, su virulencia fue estudiada mediante la infección del modelo C. elegans. En los resultados se observó que los mutantes derivados de PAOMS desarrollaron resistencia a imipenem antes que los derivados de PAO1 cuando habían sido sometidos a presión selectiva con este mismo antibiótico. Asimismo, cuando ambas cepas fueron tratadas con imipenem/relebactam, el desarrollo de resistencia frente a imipenem fue reducido. La caracterización de los mutantes mediante la secuenciación del genoma entero resaltó que los mutantes resistentes a imipenem poseían mutaciones en los genes relacionados con la hiperproducción de la cefalosporinasa AmpC y la porina OprD, en cambio, aquellos mutantes resistentes a imipenem/relebactam demostraron mutaciones en el sistema de eflujo MexAB-OprM así como también en oprD. Finalmente, el estudio de la virulencia en C. elegans demostró que aquellos mutantes más resistentes fueron menos virulentos, e inversamente los mutantes menos resistentes resultaron virulentos. Estos resultados sugieren que el relebactam es la combinación más adecuada junto con el imipenem para remediar infecciones causadas por P. aeruginosa resistente a carbapenémicos