Phenotypic and genomic changes in entericKlebsiellapopulations during long-term ICU patient hospitalization: The role of RamR regulation

Acquired antimicrobial resistance and metabolic changes are central for bacterial host adaptation during the long-term hospitalization of patients. We aimed to analyze the genomic and phenotypic evolution of enteric Klebsiella populations in long-term intensive care unit (ICU) patients. Weekly rectal swabs were prospectively collected from all patients admitted to the ICU in a teaching hospital from December 2018 to February 2019. The inclusion criterion for patients was hospitalization for more than 15 days in the ICU without any history of hospitalization or antibiotic treatment for the three months prior to admission. Among them, enteric Klebsiella pneumoniae species complex (KpSC) populations were detected. For each isolate, extensive antimicrobial resistance profiles were determined using the disk diffusion method, and the whole genome was sequenced using an Illumina platform. In silico typing methods, such as multilocus sequence typing (MLST), core-genome MLST, SNP typing, resistome characterization and mutation point detection, were applied. During the study period, 471 patients were admitted to ICUs. Among them, 21 patients met the inclusion criteria, and only five patients (24%) carried unique and distinct KpSC populations during two to ten weeks in the gut that as detected at admission and excluding acquisition during the ICU stay. One patient showed a rare ST1563 K. variicola persistent carriage for seven consecutive weeks, which displayed important antimicrobial resistance phenotype changes in the two last weeks. In-depth in silico characterization and RNA sequencing of these strains revealed a mutation within the ramR transcriptional regulator resulting in overexpression of the ramA regulator and decreased expression of acrR . These modifications are implicated in multidrug resistance, biliary salt tolerance and other bacterial functions. This study revealed the importance of endogenous colonization of KpSC populations in the gut throughout the patient’s long-term ICU stay and highlighted the role of ramR regulation in microbial adaptation. Author summary Klebsiella variicola belongs to a large bacterial complex named the Klebsiella pneumoniae species complex (KpSC). These bacteria are largely involved in nosocomial infection and are able to colonize human gut microbiota during hospitalization and/or develop antimicrobial resistance during the hospital stay. In this work, we aimed to determine the prevalence at admission and adaptation of persistent KpSC populations in the gut of long-term ICU patients. Among 471 patients admitted, 21 were hospitalized for more than 15 days, and 5 carried a unique and distinct endogenous KpSC. K. variicola was detected in two of the five patients, and antibiotic resistance was detected during long-term hospitalization in these patients. One K. variicola strain became cross-resistant to chloramphenicol, quinolones and tigecycline after the seventh week of hospitalization. In silico analyses revealed the persistence of a rare ST 1563 K. variicola population with a mutation in the ramR transcriptional regulator, which controls RND efflux pump expression and antibiotic efflux. This mutation also impacts tolerance to biliary salts and probably biofilm formation. In conclusion, a mutation in an important transcriptional regulator, ramR, could be involved in not only antimicrobial resistance but also facilitate persistent K. variicola colonization.