Novel ZnO nanoparticle activity against Liberibacter crescens growth and biofilm formation

Abstract

Huanglongbing (HLB) or citrus greening is the most devastating citrus disease worldwide and it severely affects the US citrus industry with millionaire losses annually. The phloem limited, insect vectored and uncultivable nature of its causal agent, ‘Candidatus Liberibacter asiaticus’, makes this disease to be incurable and unmanageable to the date by conventional methods. In this work, the in vitro antimicrobial activity of Zinkicide™, a novel ZnO based nanoformulation, was evaluated in batch cultures and under flow conditions, using Liberibacter crescens (Lcr) as a biological model for Liberibacter spp. Initially, cultural factors to obtain Lcr in biofilms in vitro were studied. Media optimization was performed by manipulating concentrations of methyl-β-cyclodextrin (mβc) and fetal bovine serum (FBS) in media BM7. The use of the alamarBlue® cell proliferation kit confirmed mβc stimulates Lcr viability and showed Lcr forms more biofilm in response to adverse environments. Cell adhesion force assays in microfluidic chambers (MC) and biofilm quantification in batch cultures confirmed that the BSA contained in the FBS fraction of the BM7 medium, prevents cell-surface attachment and demonstrated that Lcr-surface attachment does not rely on protein synthesis. Cell-cell aggregation assays showed Lcr aggregates more in the optimized media formulation than in BM7. Microscopic characterization of floating and attached Lcr biofilms showed both structures are embedded in an EPS extracellular matrix and that surface-attached biofilms are formed mainly on the flask bottom. Optimum conditions to assess Lcr growth and cell viability in microfluidic chamber systems were also determined. iii ZinkicideTM minimum inhibitory concentration in microtiter assays was 52ppm, while the minimum bactericidal concentration was 183ppm. When used at 104ppm, the compound inhibited more than the 96% of the biofilm formation. ZinkicideTM was not effective disrupting preformed biofilms in 96 well plates. In MC, Zinkicide™ did not disrupt initially Lcr adherent cells or disrupted preformed biofilms, but the occurrence of massive cell membrane damage on the Zinkicide™ treated main channels for both experiments was revealed by using the DEAD/LIVE Bactlight viability kit. The spatial antimicrobial activity assessment of Zinkicide™ in MC, showed this compound acts in gradients in fluidic vessels according to structural splits, flow direction, and cell concentration. In summary, in this work we described by the first time the biofilm formation process for the Liberibacter genus, and identified the cultural factors that trigger this phenome in vitro. We also evaluated the capacity of ZinkicideTM to inhibit Lcr growth and biofilm formation and discovered an unexpected resilience of Lcr biofilms against this antimicrobial compound.