Heteroresistance to amikacin in Klebsiella aerogenes isolates from patients in an intensive care unit in Brazil

Abstract

Heteroresistance is defined by the presence of subpopulations within a bacterial isolate that exhibit greater antibiotic resistance than the dominant population. In this study, we investigated amikacin heteroresistance in the opportunistic, nosocomial pathogen Klebsiella aerogenes (AHR-KA). Eight carbapenemase-resistant, but amikacin-susceptible, isolates from intensive care units of a Brazilian hospital were analyzed. Population analysis profiling identified five amikacin heteroresistant isolates (AHR-KA-1 to 5), with subpopulation frequencies ranging from 1.83 & times; 10-7 to 6.01 & times; 10-6. Among these, only AHR-KA-1 exhibited stable heteroresistance following serial passaging in antibiotic-free media. AHR-KA-1 demonstrated only slightly reduced growth rates when compared with those of the unstable AHR-KAs, parental, and control strains, suggesting no significant fitness cost associated with drug resistance. Time-kill assays for AHR-KA-1 showed an initial decline in cell viability, followed by regrowth at both 4 & times; and 8 & times; MIC. The draft genome of the stable isolate had a total length and G+C content similar to most of the sequenced genomes from K. aerogenes. Notably, AHR-KA-1 ' s aac(6 ')-Ib-cr variant contained D179Y and R102W mutations, conferring amikacin resistance. Moreover, quantitative reverse transcription PCR revealed significantly elevated expression of the aac(6 ')-Ib-cr gene in AHR-KA-1 before amikacin-free passage, compared to both the parental strain and the AHR-KA-1 strain after drug-free passage. Therefore, this study identified amikacin heteroresistance in carbapenemase-producing K. aerogenes, including a stable, mutation-driven phenotype with low fitness cost and rapid regrowth under high amikacin concentrations, underscoring its clinical relevance. These findings reinforce the importance of detecting heteroresistant subpopulations and strengthening surveillance to prevent treatment failure and the spread of undetected resistance.IMPORTANCEKlebsiella aerogenes, an opportunistic human pathogen, is frequently implicated in severe and invasive infections, particularly in immunocompromised individuals. The growing prevalence of antibiotic resistance among these strains poses a significant therapeutic challenge. The phenomenon of heteroresistance further complicates management, potentially leading to diagnostic difficulties due to the lack of standardized detection methods and subsequent treatment failures. Our studies identified and characterized K. aerogenes strains heteroresistant to amikacin, isolated from patients in an intensive care unit. Such data can serve as a foundational reference for understanding the clinical relevance, genomic variability, and pathogenic potential of K. aerogenes heteroresistance to antibiotics used in clinical settings.