Overexpression of the crp gene promotes biofilm formation and increases antibiotic resistance in bovine-derived Klebsiella pneumoniae.

BACKGROUND

The multidrug resistance of bovine-derived Klebsiella pneumoniae is a significant concern, with biofilm formation serving as a major factor in the escalation of antibiotic resistance. The function of cAMP receptor protein (CRP), which is encoded by the crp gene and acts as a central regulator of environmental sensing and virulence, remains unclear in pathogenic strains derived from livestock.

PURPOSE: This study aims to investigate the influence of CRP overexpression on biofilm formation and antibiotic resistancein bovine-derived Klebsiella pneumoniae, with a particular focus on its effect against cotrimoxazole.

METHODS

Recombinant strains with constitutive (Pkan) and inducible (Ptac) promoter-driven CRP overexpression were constructed using molecular cloning. Gene and protein expression were validated using RT-qPCR and immunoblotting analyses. Biofilm formation was quantified by crystal violet staining, antibiotic susceptibility to 23 agents was assessed using the Kirby-Bauer disk diffusion method, and metabolic burden was evaluated through growth curve analysis.

RESULT: The CRP-overexpressing strain (KAN group) showed a 2.9-fold increase in CRP protein expression (p < 0.01) and a significant enhancement in biofilm formation (p < 0.0001), without significant impact on bacterial growth. Notably, a reversal in antibiotic susceptibility was observed: while the wild-type strain was sensitive to cotrimoxazole (inhibition zone: 22 mm), the CRP-overexpressing strain displayed complete resistance (inhibition zone: 7 mm).

CONCLUSION

Overexpression of CRP protein promotes biofilm formation and confers resistance to cotrimoxazole in bovine-derived Klebsiella pneumonia, indicating that CRP-mediated biofilm formation might be a key mechanism driving the observed cotrimoxazole resistance in this strain.