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. 2019 Feb 19:10:217.
doi: 10.3389/fmicb.2019.00217. eCollection 2019.

Nanocomposite of Ag-Doped ZnO and AgO Nanocrystals as a Preventive Measure to Control Biofilm Formation in Eggshell and Salmonella spp. Entry Into Eggs

Belchiolina Beatriz Fonseca  1 , Paula Luiza Alves Pereira Andrada Silva  1 , Anielle Christine Almeida Silva  2 , Noelio Oliveira Dantas  2 , Aline Teodoro de Paula  2 , Otavio Cintra Lemos Olivieri  1 , Marcelo Emilio Beletti  3 , Daise Aparecida Rossi  1 , Luiz Ricardo Goulart  4
Affiliations

Nanocomposite of Ag-Doped ZnO and AgO Nanocrystals as a Preventive Measure to Control Biofilm Formation in Eggshell and Salmonella spp. Entry Into Eggs

Belchiolina Beatriz Fonseca et al. Front Microbiol. .

Abstract

Salmonella spp. is an important foodborne agent of salmonellosis, whose sources in humans often include products of avian origin. The control of this bacterium is difficult especially when Salmonella spp. is organized into biofilms. We hypothesized that the novel nanocomposites of ZnO nanocrystals doped with silver (Ag) and silver oxide (AgO) nanocrystals (ZnO:Ag-AgO) synthesized by the coprecipitation method could control or prevent the formation of Salmonella Enteritidis (SE) and Salmonella Heidelberg (SH) biofilm and its entry into turkey eggs. The diffraction characteristics of ZnO and AgO showed sizes of 28 and 30 nm, respectively. The Zn to Ag substitution into the ZnO crystalline structure was evidenced by the ionic radius of Ag+2 (1.26 Å), which is greater than Zn+2 (0.74 Å). For the SE analyses post-biofilm formation, the ZnO:Ag-AgO was not able to eliminate the biofilm, but the bacterial load was lower than that of the control group. Additionally, SE was able to infiltrate into the eggs and was found in both albumen and yolk. For the SH analyses applied onto the eggshells before biofilm formation, the ZnO:Ag-AgO treatment prevented biofilm formation, and although the bacterium infiltration into the eggs was observed in all treated groups, it was significantly smaller in ZnO:Ag-AgO pre-treated eggs, and SH could not reach the yolk. There was no difference in pore size between groups; therefore, the inhibition of biofilm formation and the prevention of bacterium entry into the egg were attributable to the use of ZnO:Ag-AgO, which was not influenced by the egg structure. Although the amount of Ag and Zn in the shell of the ZnO:Ag-AgO group was greater in relation to the control, this difference was not detected in the other egg components. In the search for new measures that are effective, safe and viable for controlling microorganisms in poultry farming, the application of a nanocomposite of Ag-doped ZnO and AgO nanocrystals appears as an alternative of great potential to prevent Salmonella sp biofilms in eggshells and other surfaces.

Keywords: bacterium; disinfection; nanocrystal; pore; preventive.

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Figures

FIGURE 1
FIGURE 1
(A) X-ray patterns of ZnO nanocrystals (black line) and nanocomposite (ZnO:5Ag; green line). (B) Amplification to investigate the alterations of the ZnO main peak due to the incorporation of Ag.
FIGURE 2
FIGURE 2
(A) Scanning microscopy image of nanocomposite (ZnO:5Ag), (B) EDS results, and (C) Quantitative elements in weight%.
FIGURE 3
FIGURE 3
Evaluation of the efficiency of ZnO:Ag-AgO in the manage the already formed SE biofilm. (A) Percentage of shell, albumen and yolk positive for SE submitted to different types of disinfection. Different letters represent statistical differences in the presence of SE in the shell (a,b), albumen (c,d) or yolk (e,f) of the eggs. NC: negative control; PA: Peracetic acid; PC: Positive control. (B) Bacterial load on SE biofilms formed in turkey eggs subjected to different types of disinfection. ZnO:Ag-AgO and PA caused a significant drop in the contaminant load on SE biofilms formed in turkey eggs. NC: negative control; PA: Peracetic acid; PC: Positive control.
FIGURE 4
FIGURE 4
Evaluation of the efficiency of ZnO:Ag-AgO in the manage the already formed SH and in the prevention of SH biofilm formation. (A) Percentage shell, albumen and yolk positive for SH submitted to different types of disinfection. Different letters represent statistical differences for the presence of SH in the shell (a,b), albumen (c,d) or yolk (e,f) of the eggs. (ZnO:Ag-AgO) pre and PA pre prevented the biofilm formation. (ZnO:Ag-AgO) pre: preventive treatment with nanocomposite of ZnO:Ag-AgO; PA pre: preventive treatment with peracetic acid; NC: negative control; (ZnO:Ag-AgO) post, treatment after biofilm formation with nanocomposite of ZnO:Ag-AgO; PA post: treatment after biofilm formation with peracetic acid; PC: Positive control. (B) Bacterial load on SH biofilms formed in eggshell subjected to different types of disinfection. (ZnO:Ag-AgO) pos and PA post caused a significant drop in the contaminant load on eggshell SH biofilms. (ZnO:Ag-AgO) pre and PA pre prevented the biofilm formation. (ZnO:Ag-AgO) pre: preventive treatment with nanocomposite of ZnO:Ag-AgO; PA pre: preventive treatment with peracetic acid; NC: negative control; (ZnO:Ag-AgO) post: treatment after biofilm formation with nanocomposite of ZnO:Ag-AgO; PA post: treatment after biofilm formation with peracetic acid; PC: Positive control.
FIGURE 5
FIGURE 5
Formation of SH biofilms on an eggshell in PC, (ZnO:Ag-AgO) pre and NC groups. (A) PC – Typical SE biofilm matrix formation (as seen on the white arrows), with invasion of bacteria as indicated by the black arrows. (B) PC – Biofilm of SH in stage of expansion, with visualization of the bacteria releasing the matrix. (C) PC – Biofilm of SH and presence of free bacteria on the surface. Length measurement (2,245 μm). (D) PC – Biofilm of SH and presence of free bacteria on the surface. Width measurement (703.1 nm). (E) (ZnO:Ag-AgO) pre – Structure similar to the SH biofilm matrix, however, it is unconfigured and bacteria are visualized in different formats. (F) NC – Group of cells in different formats verified near the unconfigured matrix.
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