Inactivation of Non-Enveloped Viruses and Bacteria by an Electrically Charged Disinfectant Containing Meso-Structure Nanoparticles via Modification of the Genome

Abstract

Introduction: A previous study demonstrated the virucidal effect of an electrically charged disinfectant (CAC-717), which contains meso-structure nanoparticles, on enveloped viruses (influenza viruses). However, the effect of CAC-717 on other microorganisms and the mechanisms by which CAC-717 inactivates the microorganisms remain unclear. In this study, CAC-717 was further evaluated in terms of its biocidal and virucidal activity as well as its effect on bacterial and viral nucleic acids.

Methods: The inactivation effects of CAC-717 against various microorganisms [non-enveloped virus, feline calicivirus (FCV); bacteria, Salmonella enterica and Escherichia coli] were investigated by comparing the viral titer of the medium tissue culture infectious dose (TCID₅₀) and the D value (estimated treatment time required to reduce the number of microorganisms by 90%). Furthermore, the effects of CAC-717 on viral and bacterial genomic RNA/DNA were examined using a polymerase chain reaction (PCR).

Results: Treatment of an equal volume of CAC-717 with cell lysate infected with a non-enveloped virus, feline calicivirus (FCV), reduced the TCID₅₀. Viral titer dropped below the detection limit after 2 min of treatment. The D value of FCV was 0.256 min (average of multiple endpoint D values) and endpoint D value was 0.341 min. The D value for E. coli and S. enterica was 0.290 min and 0.080 min (average of multiple endpoint D values), respectively and the endpoint D value was 0.545 min and 0.054 min, respectively. In addition, PCR showed the inhibition of nucleic acid amplification of the RNA and DNA genome of FCV and bacteria, respectively.

Conclusion: Our findings suggest that CAC-717 inactivates viruses and bacteria by modifying the viral and bacterial nucleic acids.

Keywords: E. coli; Salmonella; class I disinfectant; feline calicivirus; food safety; meso-structure.

Figure 1

Electron microscopic image of the meso-structure in CAC-717 After treatment with osmium tetroxide, CAC-717 was subjected to scanning electron microscopy (SEM) analysis (JSM-7500F; JEOL Ltd., Tokyo, Japan). The scale bar indicates 100 nm.

Figure 2

Treatment with CAC-717 reduced the viral titer of feline calicivirus (FCV). The FCV–infected cell lysate was exposed to an equal volume of CAC-717 for the indicated time (min) at 25°C. Viral titer of FCV [TCID₅₀ (median tissue culture infectious dose)/mL] was calculated after CAC-717 treatment as described in Materials and Methods. Zero for virus titer means below the detectable limit. Differences where *p<0.05 versus control (0 min) were considered significant when verified by the non-repeated measured analysis of variance (ANOVA) followed by the Bonferroni correction using GraphPad Prism7 software (GraphPad Prism Software Inc., La Jolla, CA, USA).

Figure 3

Immunofluorescent assay showed a reduction of FCV infectivity after CAC-717 treatment. The FCV–infected cell lysate was subjected to CAC-717 treatment for the indicated time (min) at 25°C. Recovered lysate was added to Candell-Rees feline kidney (CRFK) cells and after culturing for 1 day the cells were fixed with glutaraldehyde and analyzed by an immunofluorescent assay using anti-FCV antibody. Uninfected control (0 min) was included. Phase contrast images and the results of the immunofluorescent assay for FCV (Green) (exposure time 4 sec) are shown. The scale bars indicate 100 µm.

Figure 4

Decrease in viable numbers of Escherichia coli and Salmonella enterica after CAC-717 treatment. An aliquot of E. coli or S. enterica culture was mixed with an equal volume of CAC-717 and incubated for the indicated time at 25°C. After treatment, E. coli or S. enterica was resuspended in distilled water and spread on LB (Luria-Bertani) agar medium for E. coli or sheet medium (Sanita-kun; JNC Corporation, Tokyo, Japan) for S. enterica. The bacteria on the medium were then cultured for 1 day at 37°C for E. coli and 2 days at 35°C for S. enterica. The number of colonies on each plate were then counted. Colony forming units (CFU) per mL of (A) E. coli and (B) S. enterica treated with CAC-717 for the indicated time at 25°C are shown. Differences where **p<0.01 versus control (0 min) were considered significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction using GraphPad Prism7 software. Zero log₁₀ CFU/mL means below the detectable limit.

Figure 5

Damage to the bacterial genomic DNA caused by CAC-717 treatment. E. coli and S. enterica were subjected to CAC-717 treatment for the indicated time (min) at 25°C. Extracted genomic DNA was then analyzed to compare the levels of intact genomic DNA by polymerase chain reaction (PCR) using Takara bacteria 16S rDNA PCR kit Fast(800) and One shot PCR for Salmonella as described in Materials and Methods. An arrowhead indicates the internal control, while arrows indicate the amplified PCR products for 16S rDNA in E. coli and invA in S. enterica, respectively.

Figure 6

Damage to FCV genomic RNA caused by CAC-717 treatment. FCV–infected cell lysate was subjected to CAC-717 treatment for the indicated time (min) at 25°C. Extracted viral RNA was then analyzed to compare the levels of intact viral RNA by real-time PCR using primers for FCV as described in Materials and Methods. Differences where **p<0.01 versus control (0 min) were considered significant when verified by the non-repeated measured ANOVA followed by the Bonferroni correction using GraphPad Prism7 software. NS: no significant difference compared with control (0 min).