Table 1.
Oligonucleotides used in this study.
Fig 1.
Antimicrobial effect of the nanocomposites on pellicle biofilm (PB), planktonic cells, and surface-attached biofilm (SB) of V. cholerae.
Samples were exposed to different concentrations of (a) ZEO-AgNPs, (b) ZEO-CuNPs, and (c) ZEO-ZnNPs; (ZEO was used as control). Colony-forming unit per ml (CFU/ml) was determined by plate count. Treatments was performed by triplicate, values shown from a representative experiment are means (SD) of each treatment. Significant changes are marked with asterisk: * p-value < 0.05, ** p-value <0.01.
Fig 2.
Structural analysis of pellicle biofilms exposed to nanocomposites.
Scanning electron microscopy (SEM) of V. cholerae treated with (a) ZEO as a control, (b) ZEO-AgNPs (1 μg/ml), (c) ZEO-CuNPs (420 μg/ml) and, (d) ZEO-ZnNPs (420 μg/ml. Representative images of n = 3 biological replicates are shown.
Fig 3.
Structural analysis of planktonic cells treated with nanocomposites.
Scanning electron microscopy (SEM) images of V. cholerae treated with (a) ZEO as a control, (b) ZEO-AgNPs (1 μg/ml), (c) ZEO-CuNPs (420 μg/ml) and, (d) ZEO-ZnNPs (420 μg/ml). Representative images of n = 3 biological replicates are shown.
Fig 4.
Structural analysis of surface-attached biofilms treated with nanocomposites.
Scanning electron microscopy (SEM) was performed of V. cholerae treated with (a) ZEO as a control, (b) ZEO-AgNPs (1 μg/ml), (c) ZEO-CuNPs (420 μg/ml) and, (d) ZEO-ZnNPs (420 μg/ml). Representative images of n = 3 biological replicates.
Fig 5.
Relative expression of genes involved in biofilm formation after nanocomposites treatment.
(a) Pellicle biofilm (PB) and (b) surface-attached biofilm (SB), treated at 1 μg/ml of ZEO-AgNP (gray), 420 μg/ml of ZEO-CuNPs (green) and ZEO-ZnNPs (yellow). A quantitative RT-PCR assay and Livak method analysis were used. The means (SD) values are shown (n = 3). The expression of each gene with ZEO were considered as 1 (horizontal line); ZEO as the zeolite matrix without metallic nanoparticles (control). For PB and SB, dnaE expression was used as internal control gene. Significant changes are marked with asterisk: * p-value < 0.05, ** p-value <0.01.
Fig 6.
Relative abundance of the outer membrane proteins in pellicle biofilms (PB) after nanocomposites treatment.
(a) SDS-PAGE of OMPs fractionation samples of PB exposed to ZEO (control), ZEO-AgNPs, ZEO-CuNPs and ZEO-ZnNPs. The OMPs are indicated by arrowheads. (b) OMPs relative abundance of PB treated at 1 μg/ml of ZEO-AgNP (gray), 420 μg/ml of ZEO-CuNPs (green) and ZEO-ZnNPs (yellow). The abundance of each OMP with ZEO treatment was considered as 1 (horizontal line). Densitometric analysis of SDS-PAGE gels was performed with ImageJ software from 3 independent OMPs purification experiments (n = 3). Significant changes are marked with asterisk: * p-value < 0.05, ** p-value <0.01.
Fig 7.
Relative abundance of the outer membrane proteins in surface-attached biofilms (SB) after nanocomposites treatment.
(a) SDS-PAGE of OMPs fractionation samples of SB exposed to ZEO (control), ZEO-AgNPs, ZEO-CuNPs and ZEO-ZnNPs. The OMPs are indicated by arrowheads. (b) OMPs relative abundance of SB treated at 1 μg/ml of ZEO-AgNP (gray), 420 μg/ml of ZEO-CuNPs (green) and ZEO-ZnNPs (yellow). The abundance of each OMP with ZEO treatment was considered as 1 (horizontal line). Densitometric analysis of SDS-PAGE gels was performed with ImageJ software from 3 independent OMPs purification experiments (n = 3). Significant changes are marked with asterisk: * p-value < 0.05, ** p-value <0.01.