Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA

Highlights

• Combination of lytic phages and EDTA inhibited the re-growth of phage resistant bacteria.

• Chelation of Mg²⁺ by EDTA seems important for the combined effects with phages.

• Phages decreased viability of Campylobacter at low temperature and inhibited the re-growth in combination with EDTA.

Abstract

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

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.