Sub-Inhibitory Exposure to Meropenem Upregulates Virulence Gene Expression in Carbapenem Resistant <i>Klebsiella oxytoca</i>

Background: Klebsiella oxytoca (K. oxytoca) is an opportunistic Gram-negative pathogen that is becoming linked to healthcare-associated infections. The development of carbapenem-resistant strains is a major cause for concern in the clinical setting. Sub-inhibitory concentrations (sub-MICs) of antibiotics have been shown to affect the virulence and the formation of biofilms for bacteria. Objective: This study was designed to assess the effect of sub-MIC levels of meropenem on the expression of some virulence genes in carbapenem-resistant K. oxytoca. Methods: Ten clinical isolates of carbapenem-resistant K. oxytoca were isolated from patients diagnosed with hemorrhagic colitis. Isolates were determined by phenotypic, biochemical, and molecular techniques. Antibiotic susceptibility testing and minimum inhibitory concentration (MIC) determination were done based on the CLSI guidelines. Biofilm formation was measured by using the microtiter plate assay. The expression of virulence-associated genes (mrkA, fimA, pilQ, matB, and npsB) was studied by means of quantitative real-time PCR (RT-qPCR) under planktonic and biofilm growth conditions and after exposure to sub-MIC concentrations of the antibiotic meropenem. Results: All isolates were resistant to multiple drugs but sensitive to colistin, fosfomycin, and nitrofurantoin. Biofilm formation assays showed that all the isolates were moderate producers of biofilm. Gene expression analysis showed that virulence genes were significantly more expressed in biofilm than in planktonic condition, i.e., mrkA (2.6-fold), fimA (2.2-fold), pilQ (1.6-fold), matB (1.4-fold), and npsB (1.2-fold). Exposure to sub-MIC meropenem led to further expression of mrkA (1.8-fold) and fimA (1.6-fold). Conclusions: The results suggest that the expression of virulence-associated genes in carbapenem-resistant K. oxytoca can be increased by biofilm growth and sub-MIC antibiotic exposure. These results call on the potential function of sub-therapeutic levels of antibiotics in modulating the pathogenicity of bacterialpathogens.