Within-host evolution of Klebsiella pneumoniae from intestinal carriage to bacteremia

Klebsiella pneumoniae is a Gram-negative bacterium that normally resides in the human gastrointestinal tract. Gut colonization is a major risk factor for extra-intestinal K. pneumoniae infections. Yet, the mechanisms by which this opportunistic pathogen induce disseminated infection still poorly understood. To better understand this phenomenon, identical pairs-strain recovered from rectal swab and blood sample of 22 different Intensive Units Care (IUC) patients were recovered. Experimental approaches and genome within-patient evolution analyses were combined to identify major changes between isolates.K. spp strains (K. pneumoniae and K. oxytoca) used in this study were isolated between January 2017 and March 2021 from patients hospitalized in intensive care units. The K. spp strains isolated from patients who developed bacteriaemia and the strains of these same patients, with the same Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI TOF) profiling, present in their previous rectal sampling were retained and further analyzed. Only strains with similar MALDI TOF profiling but also a similar Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR amplification (Primers ERIC 1 (5'-ATG TAA GCT CCT GGG GAT TCA-3') et ERIC 2 (5'-AAG TAA GTG ACT GGG GTG AGC G-3') (24) found in the same patient in blood culture and rectal carriage were retained.The ability of these strains to form biofilm in contact with epithelial colon human adenocarcinoma cell line Caco-2 was studied by crystal violet staining.The ability of these strains to destabilize the cell junctions of Caco-2 epithelial cells in vitro was studied by measuring transepithelial electrical resistance.The antibiotic susceptibility of these strains was compared between those found in rectal carriage and those in bloodculture.Then whole-genome sequencing and sequence analyses of theses strains was performed. Each bloodstream sample was compared to its respective stool sample sequence using snippy using the --cntg option. For consistency, the blood samples were also compared to their respective bloodstream samples. To verify these results and further analyse genome rearrangements or large deletions or unmapped sequences, we also used breseq with default parameters.. Kleborate (v2) was used to determine sequence type, K and O locus, and characterize the different isolates in terms of virulence factors. To detect and annotate antibiotics resistance genes (ARGs), we used ResFinder v4.6. Macsyfinder v2 and the package T6SS was used to identify the presence of Type 6 secretion systems.A phylogenetic tree was built to compare the different strains, and mobile genetic elements were characterized.In summary, this work illustrates within-host evolution, during multiple antibiotics treatments, of a major nosocomial pathogen which is mostly driven by horizontal gene transfer. Because isolates were initially chosen by their ability to resist to antibiotics, future research will have to be conducted on initially susceptible strains and extend to different samples from disseminated infection in the same patient (bronchoalveolar lavage, urine or medical device). Providing a better understanding of the evolutionary capacity of bacteria are important for predicting within-patient antimicrobial resistance emergence and developing strategies of pathogens surveillance and infection control.