Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae

Abstract

Introduction: Klebsiella pneumoniae is an opportunistic pathogen that causes a range of diseases. The appearance of extended-spectrum β-lactamase -and carbapenemase-producing strains, in addition to the biofilm-forming phenotype, is a major problem in the clinical environment.

Methodology: A total of 33 clinical K. pneumoniae isolates were used in this study. Antimicrobial susceptibilities were assessed by a disc diffusion assay. Biofilm formation was determined by a microtiter plate assay, staining with 1% crystal violet and measuring  absorbance after destaining. Moreover, expression of acrA, kdeA, ketM, kpnEF, and kexD efflux associated genes  was measured by qRT-PCR.

Results: Isolates displayed high resistance to β-lactams such as cefazolin, cefuroxime, ceftriaxone, cefepime, piperacillin-tazobactam, imipenem, and meropenem and decreased resistance to gentamicin, amikacin, ciprofloxacin, and levofloxacin. ESBL-producing isolates formed more biofilm than carbapenemase-producing isolates. The mRNA expression levels in KPC isolates for acrA (2-fold), kdeA (2.7-fold), ketM (2.2-fold), and kpnEF (3.4-fold) were significantly increased compared to ESBL-producing isolates. There was no significant difference in kexD expression level.

Conclusions: Under the conditions used here ESBL-producing isolates formed more biofilm than KPC postive isolates; this was associated with virulence determinants which were also transferred by plasmids together with ESBLs enzymes. Moreover, the upregulation of acrA, kdeA, ketM, and kpnEF efflux pumps was seen in carbapenemase-producing isolates demonstrating that high expression of efflux pumps alone could not confer resistance but may act as a physiological determinant such as bacterial pathogenicity and virulence, and cell-to-cell communication for bacteria.