Autoaggregation-associated carbapenem tolerance mediated by mrkH inactivation identified from a cohort of Klebsiella pneumoniae species complex clinical isolates

OBJECTIVE: The objective of this study is to elucidate the phenotypic and genetic basis of carbapenem tolerance in Klebsiella pneumoniae species complex bloodstream isolates lacking carbapenemase genes, with a focus on multicellular behaviours. MATERIALS AND METHODS: Fifty clinical K. pneumoniae species complex isolates from bloodstream infections were screened for reduced susceptibility to meropenem. Five strains (MIC 1-2 mg/L) lacking carbapenemase genes were analysed using whole-genome sequencing, time-kill assays, plasmid-based gene expression assays and phenotypic assays, including autoaggregation and biofilm formation. RESULTS: The results of the time-kill assay showed that three strains were carbapenem tolerant. Two strains carrying blaDHA-1 exhibited reduced meropenem activity and enhanced survival following drug exposure. One isolate showed sustained survival and carried a frameshift mutation in mrkH, leading to downregulation of mrkA expression and enhanced autoaggregation. Complementation with wild-type mrkH reduced tolerance. Disruption of cellular aggregates significantly decreased survival, indicating that aggregation provides physical protection against meropenem. CONCLUSIONS: This study identified mrkH-linked autoaggregation as a novel mechanism of carbapenem tolerance in Klebsiella isolates. Overall, the findings underscore the role of multicellular behaviours in antibiotic resilience and highlight the potential clinical relevance of non-carbapenemase-mediated tolerance mechanisms.