Abstract
Background: K. pneumoniae is capable of resistance to β-lactam antibiotics through expression of β-lactamases and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. β-lactamase expression has long been presumed to have a previously unidentified fitness cost. In fact, little is known about how β-lactamase expression affects cellular physiology and whether these fitness costs may relate to other resistance or virulence mechanisms. The purpose of this dissertation was two-fold. First, to determine the extent to which β-lactamases and outer membrane porins affected β-lactam resistance. Second, to evaluate how β-lactamase carriage altered global RNA transcription in the cell and influenced resistance and virulence factors.Methods: Outer membrane porins were evaluated by rt RT PCR and western blot of both whole cell lysates and outer membrane fractions. β-lactamase carriage was determined by whole genome sequencing and expression evaluated by rt RT PCR. MICs to cefotaxime, ceftazidime, cefepime, ceftolozane/tazobactam, ceftazidime/avibactam, meropenem, meropenem-vaborbactam, and imipenem-relebactam were determined by E-test using CLSI guidelines. β-lactamase clones were generated by restriction digest cloning. RNA-sequencing of β-lactamase clones and bioinformatics were performed by SeqCenter. Differential expression of genes was determined by comparison of all clones to respective empty vector controls. Expression of select genes was confirmed by rt RT PCR. Biofilm production was evaluated by crystal violet assay. Adhesion was evaluated by adherence of bacterial cultures to T24 cells.
Results: Important determinants for β-lactam resistance were antibiotic dependent. Plasmid-encoded β-lactamase carriage was important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHVs was important for ceftolozane/tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. When determining the effect of β-lactamase expression on the cell, alterations of outer membrane pathways were noted, including in transport of the outer membrane porins. CTX-M-15 expression was associated with differential expression of genes in the Rcs, Cpx, and σE regulon. Removal of the CTX-M-15 signal sequence altered the transcriptomic profile and led to upregulation of cytoplasmic chaperones but did not fully ameliorate the effect of the β-lactamase. Biofilm formation was decreased when CTX-M-15 was expressed, in a protein signal sequence dependent manner. Adhesion to T24 cells was decreased when the CTX-M-15 signal sequence was removed.
Discussion: This work demonstrates that β-lactam resistance is multifactorial in K. pneumoniae. Expression of the β-lactamases influences cellular physiology resulting changes in outer membrane proteins, including OmpK35, fimbriae associated with biofilm formation and adhesins. Given altered expression of chaperones and stress response proteins, occupation of protein folding pathways by the β-lactamase likely affects overall physiology. This dissertation reveals a link between β-lactamase expression and other resistance or virulence factors through competition for protein folding pathways.