Like other lactic acid bacteria (LAB),
Methods to improve the effect of biocides on viruses by using complex treatments, such as chemical inactivation with UV radiation, synergistic chemical inactivation, and synergistic inactivation with UV and ozone, have already been reported. Pujato et al. (2014) found that 0.15% peracetic acid and 600–800 ppm of sodium hypochlorite had a good inactivation effect on
TIII: The treatment time was the same as TII, but the inactivation sequence of 75% ethanol and isopropanol, sodium hypochlorite, or peracetic acid solutions at different concentrations, as mentioned above, was reversed, respectively.
As one of the most used biocides, ethanol has been reported to destroy or denature enzymes and microbial proteins in cell walls. The ethanol concentration, similarly to most biocides, determines whether its action is biocidal or static. Low (<20%) concentrations will limit its effect and are usually biostatic (Kim et al. 2020). Moderate concentrations (60–85%) can quickly solidify microbial proteins resulting in a biocidal effect (Sauerbrei 2020). However, when the concentration is too high (>90%), it can form films on the microbial surface, thereby influencing its effect (Setlow et al. 2002; Zhang et al. 2012).
Concerning phages, biocides affect the protein structure in the capsid (Sato et al. 2016). It was reported that ethanol and isopropanol could denature proteins, which is destructive to most viruses (Maillard 2002; Mahony and Sinderen 2015; Boyce 2018). However, the previous studies found that neither ethanol nor isopropanol could completely inactivate phage P1 and P2, even after a 60 min treatment. Nevertheless, the inactivation effect of isopropanol was slightly better than that of ethanol (Chen et al. 2017; 2018). To explore whether isopropanol and ethanol could mutually improve the inactivating effect on phages, the survival counts of
Viable count of phages treated with 75% ethanol and isopropanol.
a) Treatment with isopropanol for 10 min; b) treatment with 75% ethanol and isopropanol for 10 min; c) treatment with 75% ethanol for 5 min followed by isopropanol for 5 min; d) treatment with isopropanol for 5 min followed by 75% ethanol for 5 min.
In dairy industries, sanitization between fermentations is a critical step in controlling the phage contamination (Hayes et al. 2017). It employs purpose-made chemical sanitizers for the physical and chemical removal of phages and other microbial contaminations (Pujato et al. 2018). For biocides to be considered eligible to use in the dairy industry, several criteria must be met. For example, in Europe, sanitizers must have a proven ability to reduce phage numbers by at least four logs under the recommended test conditions before they can be deemed suitable for phage inactivation (European Committee for Standardization (CEN), CEN/TE-216) (Bolten et al. 2022).
As illustrated in Fig. 1, combined or subsequent treatments with ethanol and isopropanol could not completely inactivate phages P1 and P2. However, isopropanol could enhance the biocidal effect of 75% ethanol to a certain extent. Ethanol (75%) or isopropanol (100%), when used alone for 10 min, reduced P1 by 2.45 and 2.46 log (Fig. 1a), respectively. However, when phages were treated with a mixture of 100% isopropanol and 75% ethanol for 10 min, a 3.83 log reduction was obtained (Fig. 1b). Similar results were observed for phage P2, as the treatment with ethanol (75%) for 5 min, followed by the treatment with isopropanol (100%) for 5 min, was even more effective, resulting in a 3.55 log reduction (Fig. 1c).
Both ethanol and isopropanol have been reported to destroy most lipophilic viruses (Maillard 2002; Mahony and Sinderen 2015). Although isopropanol (100%) can enhance the effect of ethanol (75%) on phages P1 and P2, the augmenting effect is limited. As reported, isopropanol can interact with microorganisms in aqueous solutions to form (CH3)2CHOH(H2O)n, or isopropanol water clusters, where the n value represents the amount of H2O molecules. The structures of water clusters formed by isopropanol in various electrostatic environments are different, providing differences in the structural stability of water clusters, thus having diverse effects on microorganisms (Han et al. 2017; Zhu et al. 2019). Different phages will produce different electrostatic environments in the solution. The charge density is a significant characteristic of each phage, which mainly depends on the nature of the phage capsid or tail protein and the difference in genetic material (Hernando Pérez et al. 2015; Cooper et al. 2022). In our previous research, the morphology of phages P1 and P2 were similar, but their structures were slightly different. The tail fibrin (CDs18) of phage P1 was different from that encoded in phage P2’s genome (Guo et al. 2022). The two electrostatic environments caused by different tail fibrin may enable isopropanol to form isopropanol-water clusters with different stability in aqueous solution, thus affecting the biological efficacy of isopropanol on phages P1 and P2.
Viable count of phages treated by 75% ethanol and sodium hypochlorite.
a) Treatment with sodium hypohlorite for 10 min; b) treatment with 75% ethanol and sodium hypohlorite for 10 min; c) treatment with 75% ethanol for 5 min followed by sodium hypohlorite for 5 min; d) treatment with sodium hypohlorite for 5 min followed by 75% ethanol for 5 min.
The phage treatment with 400 ppm of sodium hypochlorite for 10 min resulted in a 0.89 log reduction for phage P1 (Fig. 2a). As expected, the number of phage P1 survivors decreased significantly (
Sodium hypochlorite is a highly effective sanitizer. However, the efficacy of sodium hypochlorite is affected by several factors, such as pH, temperature, and organic matter. Lowering the pH, increasing the temperature, and reducing the organic load increase its antimicrobial effect (Cai et al. 2016). Hypochlorite acts directly on DNA. In this respect, it has been shown that
Sodium hypochlorite hydrolyzed in water form hypochlorous acid, which is lethal for most microbes. It has been demonstrated to interfere with carbohydrate metabolism, oxidize protein, and target phosphate dehydrogenase (Cho et al. 2010; Sato et al. 2016). Sodium hypochlorite also inactivates
As reported, 100–200 ppm sodium hypochlorite had a good inactivation effect on some phages, such as
Genomic differences between phages P1 and P2 may account for the variation in resistance. As reported, sodium hypochlorite can inactivate phages by acting on their protein structures or genome. It can cause the inactivation of coliphage MS2 through genome damage. In contrast, the inactivation
Our previous studies have shown 11 gene differences between these two phages, including eight putative proteins, one tail fibrin, and two HNH endonucleases. Moreover, phage P2 encoded more putative proteins with unknown functional gene sequences (Guo et al. 2022; Zhu et al. 2022). The difference in protein structure and the gene sequence coding proteins may be why phages P1 and P2 have different tolerance to sodium hypochlorite. Similar to our study, Briggiler Marcó et al. (2009) reported that sodium hypochlorite (800 ppm) could completely inactivate phage B2 of
Interestingly, they also had tail fibrin and different HNH endonucleases. So, genomic differences may influence the tolerance of phages to chemical biocides treatments. Phages isolated from the identical source and the same host might exhibit different tolerances to sodium hypochlorite. Inactivation effects could mainly depend on the nature of the phages (Briggiler Marcó et al. 2009).
As shown in Fig. 3, PAA was not an effective biocide against phages P1 and P2, even at a concentration of 0.45%. Phages P1 and P2 treated with the solution at a concentration of 52.6% and 63.8%, respectively, remained viable after a 10 min-treatment. Moreover, PAA did not increase the biocidal effect of ethanol (75%). As shown in Fig. 3, even the highest concentration of PAA resulted in the lowest survival rate (TII) of 2.21%. Phage P2 appeared to exhibit a higher tolerance to PAA than P1, which still exhibited a survival rate of 6.33% under the same treatment. In contrast, ethanol (75%) increased the phagocidal effect of PAA.
Viable count of phages treated by 75% ethanol and peracetic acid (PAA).
a) Treatment with PAA for 10 min; b) treatment with 75% ethanol and PAA for 10 min; c) treatment with 75% ethanol for 5 min followed by PAA for 5 min; d) treatment with PAA for 5 min followed by 75% ethanol for 5 min.
Peracetic acid exerts a strong oxidizing effect on proteins. It exerts a rapid bactericidal effect on various microorganisms, including spores and viruses (Yeap et al. 2015; Zonta et al. 2016). The treatment with peracetic acid (0.15%) for 60 min had little effect on the survival of P1 while increasing the concentration to 0.45% for 60 min resulted in only a 4.0 log reduction in the number of phages (Chen et al. 2017). Phage P2 demonstrated greater resistance to peracetic acid than P1. At the highest concentration evaluated (0.45%), the viability of phage P2 decreased by only 1.40 log within 60 min (Chen et al. 2018). Ethanol may accelerate the destruction of viruses by PAA, enhancing the mobility of PAA molecules in the disinfectant solutions. The combination of PAA (0.2%) and ethanol (80%) could result in a 4.0 log reduction of the poliovirus type 1 number in 1 min. A comparable virucidal effect could not be achieved with 80% ethanol, even if the exposure time was prolonged to 30 min (Wutzler and Sauerbrei 2000). However, like in the previous research results, PAA might not have a good bactericidal effect on phages P1 and P2, even when used with ethanol. In this respect, only phages with sulfur-containing amino acids (such as cysteine and methionine) in their capsid proteins show strong sensitivity to PAA (Schmitz 2021). The number and types of sulfur-containing amino acids in the capsid proteins of phages P1 and P2 must be further determined. In addition, differences in the protein configuration in the capsid brought about by secondary, tertiary, and quaternary folding may expose the different and sensitive abilities to the action of PAA (Mayer et al. 2015). The present study found that when PAA was mixed with ethanol (75%), the biocidal effect was not improved even at the highest concentration (0.45%).
In contrast, ethanol (75%) improved the biocidal effect of PAA, especially for P2. During the first 5 min of application, ethanol (75%) impacted the protein capsid allowing PAA more accessible contact with sulfur-containing groups. However, the present results also demonstrated that with the increase in PAA concentration, the biocidal effect was not increased significantly. Phages P1 and P2 may contain less PAA-sensitive target sites.
The dual inactivating effects of various biocides on phages P1 and P2 were evaluated. Results showed that the phages could be completely inactivated in 10 min after being treated successively with 75% ethanol and 400 ppm of sodium hypochlorite. Compared to a single biocide treatment, successive treatments could reduce the biocide’s concentration and shorten the inactivation time. This study might provide some basis for controlling phage infections in laboratories and dairy plants.