Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA
Introduction
Despite constant efforts to reduce viable counts of foodborne pathogens from farm to consumer, cross-contamination during processing, storage, and distribution are currently a foodborne illness risk (Carrasco et al., 2012). For example, the major foodborne pathogens in fresh meats and other foods include Gram-negative bacteria such as Campylobacter spp., Salmonella spp., and pathogenic E. coli, and Gram-positive bacteria such as L. monocytogenes may result in outbreaks with severe mortality (Heredia and García, 2018). Furthermore, the development of resistance to currently available antibiotics by these pathogens has attracted attention because of the severity of bacterial infections in food and water systems (Doménech et al., 2015). Hence, the use of adequate alternatives to antibiotics is an extremely emerging concern.
As the development of novel antibiotics is limited, applying new, highly effective tools to control bacterial pathogens is of paramount importance. Phages are known bacterial viruses that specifically infect and lyse bacterial cells. Interest in their use as novel biocontrol agents for food safety has increased considerably (Lin et al., 2017). The efficacy of many conventional antibiotics requires active bacterial growth. Conversely, phages possess significant advantages to control metabolically active and inactive bacteria even at refrigerated temperature (Hudson et al., 2005). In addition, phages have been shown to self-proliferate in host bacteria and to be effective in vivo and in vitro against target bacteria (Payne and Jansen, 2000). Previous studies have demonstrated that phages successfully eliminated Campylobacter (Bigwood et al., 2008), Salmonella spp. (Duc et al., 2018), E. coli O157:H7, extended-spectrum beta-lactamase-producing E. coli (Son et al., 2018), L. monocytogenes, Pseudomonas fragi, and Staphylococcus aureus (Greer, 2005) contamination in foods. However, the phage-resistant bacterial populations' emergence and re-growth are not readily eliminated and thus impose further difficulties in phage therapy (Kortright et al., 2019). Since phage adsorption to cell receptors is the first step of infection, commonly bacterial host strains could develop resistant strategies to block the specific adsorption of phages by receptor changing, competitive inhibitor production or extracellular matrix production (Chan et al., 2013). Therefore, the use of phage cocktail composing of distinct phages, which could broaden host range of phage treatment, has been promisingly proposed as a solution for minimizing the risk of development of phage resistance. However, some publications have previously reported that phage cocktails failed to inhibit the regrowth of bacterial resistant cells or was not better than single phage (Duc et al., 2020). Other approaches that have been explored to overcome phage resistance include genetic engineering, which can be a time- and resource-consuming process, as well as the combination with safe antimicrobial agents, such as bacteriocins, natural polyphenols, and chelating agents, which might be a promising alternative in food industry (Chawla et al., 2006).
Ethylenediaminetetraacetic acid (EDTA) and Ethyleneglycoltetraacetic acid (EGTA) raise concerns due to their capacity to induce chelation of divalent ions (Hülsmann et al., 2003). It is well known that EDTA damages the outer membrane permeability of Gram-negative bacteria by chelating divalent cations in the lipopolysaccharide (LPS), thus causing leakage of LPS and increasing permeability to other antimicrobials (Stevens et al., 1992). In addition, EDTA has a stronger affinity for Mg2+ ions and binds to metals via four carboxylate and two amine groups, whereas EGTA for Ca2+ (Sousa et al., 2005; Sayin et al., 2007). Unlike other conventional antimicrobials, EDTA is an effective defense against some multiple drug-resistant strains because high magnesium levels are thought to be essential for bacterial replication (Hamoud et al., 2014). EDTA also possesses a much broader spectrum of antibacterial activity than phages when applied with antibacterial agents inhibiting biosynthesis. Moreover, the health risks of EDTA has been independently assessed by the World Health Organization and the Food and Drug Association, it is approved for use in a variety of food products at concentrations ranging from 36 to 500 ppm (Heimbach et al., 2000).
Since the antibiotic-resistant bacteria are difficult to control by single antimicrobial agents, a combination of physical, chemical, and biological antibacterial methods, also called hurdle technology, is posited to effectively inhibit the growth of bacteria (Leistner, 2000; Chawla et al., 2006). Currently, few studies have demonstrated increased effectiveness with a combination of conventional antimicrobials against Gram-negative bacteria (Gómez et al., 2011; Khan et al., 2017). Thus, the potential synergistic antimicrobial effect of phages and EDTA should be considered. However, this combination has not been investigated since divalent metal ions are required to stabilize phage activity (Thorne and Holt, 1974; Whang et al., 1996).
The purpose of this study is to investigate the efficacy of EDTA in conjunction with phages to control the pathogenic bacterial growth and re-growth of phage-resistant populations. Thus, EDTA's effects on the lytic activity of several phages specific to different foodborne pathogens were investigated. To the best of our knowledge, this study is the first report to highlight the significant effect of hurdle technology consisting of phages combined with EDTA against common foodborne pathogens.
Section snippets
Bacterial strains
The four bacterial strains used in this study are listed in Table 1. S. Enteritidis NBRC 3313 and S. Typhimurium NBRC 12529 were obtained from NITE Biological Research Center (NBRC), Chiba, Japan. C. coli CAN10 strain was isolated and identified at the Fukuoka City Institute of Health and Environment, Fukuoka, Japan (Huang et al., 2021). The C. jejuni L26 strain was isolated and identified in the Food Hygienic Chemistry laboratory at Kyushu University, Fukuoka, Japan (Furuta et al., 2016). Both
Effect of chelating agents on the viability of Campylobacter in the presence of specific phages
In preliminary experiments on the combined effects of food additives and phages, EDTA effectively repressed the re-growth of C. jejuni treated with phages (data not shown). The effects of EDTA and EGTA on the viability of C. jejuni in the presence of phages were investigated (Figs. 1 and 2). In the control, C. jejuni grew and reached approximately 10 log CFU/mL after 24 h of culture. In the presence of 1.0 mM EDTA, the viable C. jejuni counts did not increase until 12 h of incubation but
Discussion
In a preliminary study, we found that the appropriate concentrations of EDTA inhibited the re-growth of phage-resistant bacterial populations without inhibiting phage lytic activity. The growth of the Gram-negative bacteria tested was slightly suppressed by EDTA alone at 0.5–1 mM (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5). In combination with phages, EDTA inhibited the re-growth of the phage-resistant C. jejuni populations at 1 mM (Fig. 1A). However, another chelator, EGTA, with a higher affinity
Conclusion
The combinations of EDTA and specific lytic phages inhibited the re-growth of phage-resistant cells of C. jejuni, C. coli, S. enteritidis, and S. typhimurium. To the best of our knowledge, this is the first study highlighting that the combined use of phages and EDTA effectively inhibits the re-growth of phage-resistant bacteria.
Declaration of competing interest
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by JSPS KAKENHI (Grant Number JP19H02912).
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