Phenotypic and genomic changes in enteric Klebsiella populations during long-term ICU patient hospitalization: the role of RamR regulation

Abstract

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 3 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 5 patients (24%) carried unique and distinct KpSC populations during 2-10 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 7 consecutive weeks, which displayed important antimicrobial resistance phenotype changes in the 2 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, which controls antibiotic efflux. This mutation also impacts tolerance to biliary salts. 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 in drug susceptibility.

Importance: The Klebsiella pneumoniae species complex (KpSC) is one of the major causes of nosocomial infections, especially in intensive care unit (ICUs). These bacteria are frequently highly resistant to antibiotics, leading to an increase in morbidity and mortality. The origins of multidrug-resistant KpSC strains isolated from ICU patients are still unclear, with at least two hypotheses of acquisition paths: (i) endogenous KpSC populations that are or became resistant to antibiotics and/or (ii) hospital acquisition of circulating KpSC clones. Genomic changes observed in this study might reveal mechanisms to better adapt to KpSC in the patient's gut in the face of heavy ICU medical care pressure.

Keywords: Klebsiella variicola; RamR; host adaptation; intensive care units; long-term carriage; population dynamic.

Fig 1

Study flow chart of patient inclusion from ICUs at Caen University Hospital Center. KpSC, K. pneumoniae species complex; n, number of patients.

Fig 2

Patient hospitalization timelines. Blue, positive; yellow, negative; dashed, decreased antibiotic susceptibility. ICU, intensive care unit.

Fig 3

Bar chart of relative gene expression between strains with functional ramR and mutated ramR. Differential gene expression between strains group ramR wildtype (ESSO2 to ESSO6) and strains group ramR mutant (ESSO7 and ESSO8) by quantitative PCR. Fold-change was calculated using ∆∆Ct method with gyrA as housekeeping gene and P values were estimated with Student’s test. Error bars represent standard deviation. Results were considered significant if fold-change more than 2 and P value less than 0.05 and represented by star.

Fig 4

Differential gene expression between isolates unmutated and mutated ramR K. variicola and gene set enrichment analysis via KEGG Ontology. (A) MA plot of differential genes expression between not mutated ramR K. variicola (esso6) and K. variicola with mutation in ramR (ESSO7). Colored points represent genes with a significant P value adjusted with Benjamini & Hochberg method ( P value adjusted < 0.05), blue point for genes overexpressed and yellow points for genes under expressed. Dashed lines represent the threshold of Fold Change. (B) Gene enrichment distribution by KEGG Ontology and (C) gene ratio of enriched pathways between the ESSO6 and ESSO7 strains. P value adjusted with the Benjamini & Hochberg method (R package “enrichplot”).