Efflux-Mediated Synergy and Preserved in Vitro Selectivity of Zinc Oxide Nanoparticle–Ampicillin Combinations against Multidrug-Resistant Klebsiella Pneumoniae and Pseudomonas Aeruginosa
Abstract
Background: Zinc oxide nanoparticles have the ability to rescue the β-lactam activity against multidrug resistant Gram-negative pathogens, but only concentrations that synergy that do not harm the host cells are therapeutically useful.
Objectives: To quantify the synergy between ZnO-NP and ampicillin in clinical isolates of MDR Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as to parallelly determine if the synergistic concentrations are selective to the bacteria compared with the human lung fibroblasts and keratinocytes.
Methods: One hundred and forty-five non-duplicate clinical isolates (71 K. pneumoniae, 74 P. aeruginosa) recovered at Fallujah General Hospital, Anbar, Iraq, were studied. MICs and MBCs were obtained using broth microdilution and synergy was evaluated by checkerboard assays and by the fractional inhibitory concentration index, dose-reduction index, ZIP, Bliss, Loewe and highest-single-agent models. Two-way mixed-design ANOVA with Greenhouse–Geisser correction and Holm adjusted paired contrasts were used to compare normalised growth under six conditions. Dose–response curve with MTT was fitted to four-parameter logistic model and the selectivity index was calculated for WI-38 and HaCaT cells. Mechanistic endpoints included ethidium bromide retention, membrane permeability, qPCR of acrA, acrB, mexA and mexB and molecular docking. Biological inclusion levels of low dose hormesis (0.2%, 0.4%, and 0.8% ZnO MIC) were tested on the biofilm biomass, metabolic activity and extracellular polymeric substances (EPS) as well as the markers of cytotoxicity, LDH release, apoptosis and necrosis, and mitochondrial membrane potential.
Results: Baseline susceptibility was closely matched between species (median ampicillin MIC 256.00 µg/mL, IQR 128.00–512.00, P = 0.386; median ZnO MIC 62.50 µg/mL, IQR 31.25–125.00, P = 0.825). Synergy occurred in 133/145 isolates (91.7%): 66/71 K. pneumoniae (93.0%) and 67/74 P. aeruginosa (90.5%), with median FICI values of 0.391 and 0.359 and a median 8-fold ampicillin dose reduction in both species. Combination growth fell to 24.26 ± 11.33% of control versus 98.76 ± 0.70% in untreated controls (partial η² = 0.974 for treatment; no species effect, P = 0.931). On WI-38 and HaCaT cells, the combination IC₅₀s were 558.8 and 613.0 µg/mL (K. pneumoniae) and 542.8 and 598.2 µg/mL (P. aeruginosa), respectively, while ampicillin alone could not be determined in the 0–1000 µg/mL range. Median SI values were 38.57–43.27 and 41.52–44.19; SI > 1 occurred in 97.2% and 94.6% of combinations. EtBr retention (1.94-1.96-fold), membrane permeability (1.59-1.64-fold), pump-gene repression (0.66-0.69-fold) and docking scores (−6.3 to −6.4 kcal/mol) all pointed towards an efflux/permeability mechanism. Host-cell injury remained low (LDH 10.2–10.3%; apoptosis 6.9–7.4%; necrosis 3.3–3.4%; MMP 87.9–89.4% of control). But, 0.2-0.8% of the ZnO MIC enhanced the formation of biofilm, metabolic activity and EPS up to 109% of control. Conclusions: The results demonstrate that the combinations of ZnO-NP/ampicillin are synergistic and selective for both species in vitro, with the combination showing a narrow sub-inhibitory exposure, so that there is a need for dose-controlled and mechanism-informed development rather than low dose application.
Keywords: Zinc oxide nanoparticles; ampicillin; efflux-mediated synergy; selectivity index.
