in vitro antibacterial activity of nordihydroguaiaretic acid combined with ceftriaxone against carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVE

This study investigated the interplay between biofilm formation and efflux pumps in carbapenem-resistant Klebsiella pneumoniae (CRKP) and evaluated the biofilm inhibition by nordihydroguaiaretic acid (NDGA) along with the efficacy of its combination with ceftriaxone (CRO) against CRKP, to inform the development of novel therapeutic strategies against multidrug-resistant infections.

METHODS

Efflux pump genes were detected via PCR. Biofilm formation was quantified by crystal violet staining and categorized by the biofilm formation index (BFI) classification. Strong biofilm producers (BFI > 4 × negative control) were selected for in vitro models. Efflux pump gene expression was compared between biofilm and planktonic states using qRT-PCR. NDGA's biofilm inhibition and NDGA-CRO synergy were assessed via checkerboard assays.

RESULTS

Among the 86 CRKP isolates, 51.16% (44/86) were classified as strong biofilm producers, followed by moderate (17.44%, 15/86), weak (20.93%, 18/86), and negative (10.47%, 9/86) producers. Efflux pump genes (acrA, acrB, tolC, oqxA, oqxB) were detected in 100%, 100%, 100%, 97.7%, and 98.84% of isolates. Gene expression was significantly upregulated in biofilm versus planktonic state (P < 0.05). NDGA (≥16 µg/mL) inhibited biofilm formation, and its combination with CRO exhibited additive (63.64%) and synergistic (36.36%) effects.

CONCLUSION

Efflux pumps and biofilms synergistically enhance CRKP resistance. By inhibiting both efflux-mediated antibiotic extrusion and biofilm formation, NDGA resensitizes CRKP to CRO. This in vitro efficacy supports further development, pending in vivo safety studies in animal models.