The role of relA-mediated stringent response on the nutritional, environmental, antimicrobial resistance and biofilm formation in Klebsiella pneumoniae and its effect on Drosophila melanogaster survival

The stringent response has far-reaching consequences, with links to antimicrobial resistance, stress and virulence. This study assessed the role of relA in nutritional, environmental and antimicrobial stress in Klebsiella pneumoniae, the extent of polysaccharide capsule and biofilm formation, and the pathogenic effect on Drosophila melanogaster. Two single mutants (KP03DrelA and KPPR1DrelA) obtained using the Lambda Red Recombinase Technique were the focus of analyses. We assessed environmental (ethanol, osmotic, heat) and nutritional stress (carbon, phosphate, amino acid) tolerance, capsule formation, and cell size determination in wild-type (WT) and mutant bacteria. Biofilm and agar plate susceptibility assays were performed on both starved and non-starved strains using gentamicin and ceftazidime, and stress response genes were analyzed. Drosophila melanogaster was used to examine the pathogenic effect of the presence or deletion of relA on fly survival. DrelA mutants had reduced fitness to environmental and nutritional stress compared to WT strains. Mutant strains cell lengths were elongated and lacked a capsule versus WT and non-starved strains, and mutant strains exhibited enhanced biofilm formation or survival in the presence of ceftazidime and gentamicin. Stress response genes rpoD, phoR, phoU and pstB were absent during starvation, but rpoS was detected in C-, C+PO+ and serine hydroxamate (SHX) media; mrkA gene was not detected during starvation. Lastly, the animal model proved to be effective in showing infection levels associated with the presence of the relA gene.