Abstract

Background and Objective: Fresh chicken meat includes the capacity to contain foodborne pathogens. A previous study has demonstrated efficacy of recombinant AGAAN antimicrobial peptide against various bacterial strains. In general, AGAAN is a newly discovered antimic-robial peptide with a unique cationic alpha-helical structure. The peptide is originated from the skin secretions of Agalychnis annae. This peptide showed a significant affinity towards the negatively-charged microbial lipid bilayer, as previously demonstrated by the experimental and in-silico analyses. However, the major concerns include high production costs, limited expression, laborious process and potential toxicity associated with concentrated peptides. In this research, the synergistic effects with organic acid were addressed to decrease these problems while preserving its bactericidal activity.

Material and Methods: Recombinant AGAAN and organic acids were assessed on Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739. This was carried out by assessing minimum inhibitory concentration and fractional inhibitory concentration. In addition, effects of the combination on bacterial membrane integrity by carrying out beta-gala-ctosidase assessment. Additionally, the potential efficacy of this combination in preserving poultry meat was investigated.

Results and Conclusion: Minimum inhibitory concentration of the recombinant AGAAN against the two bacterial strains was 0.15 mg.ml^-1. In contrast, the minimum inhibitory concentration of acetic acid against Staphylococcus aureus and Escherichia coli were 0.2 and 0.25% v v^-1, respectively. The combination demonstrated significant synergy, as evidenced by fractional inhibitory indices of 0.375 against the two foodborne pathogens. Based on the study, the combination effectively inhibited proliferation of these disease-causing microorganisms that led to foodborne illnesses within 300 min. Presence of intracellular beta-galactosidase indicated that the combination of factors has caused damages to the cell membrane, resulting in its compromised integrity. Red blood cells exposed to various concentrations of recombinant AGAAN and acetic acid did not result in hemolysis. Results showed significant differences (p < 0.05) in all the experiments on meat samples that received treatments with recombinant AGAAN and acetic acid. The current study detected that a combination of recombinant AGAAN antimicrobial peptide with organic acid could effectively inhibit growth of pathogens at lower concentrations. Data presented in this study can help food industries develop further efficient cost-effective antimicrobial uses.

1. Introduction

Prevalence of foodborne diseases has emerged as a significant global health concern. The World Health Organization (WHO) report indicates that nearly 600 million cases of foodborne illnesses occur annually due to the consumption of food substances contaminated with microorganisms and chemicals [1]. Food contamination and increases in the risk of foodborne diseases are caused by pathogenic microorganisms [2]. Meat and meat products are important sources of nutrients for humans due to their high protein composition and other essential nutrients [3].  However, these foods provide appropriate environments for the growth of foodborne microbes due to their high water content and nutrients, [4]. A significant number of studies have shown that Staphylococcus aureus and Escherichia coli are associated with meat contamination [5-7]. The S. aureus is a facultative anaerobic, Gram-positive non-spore-forming bacterium [8]. It is a major problem in foodborne illnesses [9]. The S. aureus infections cause significant morbidity and mortality in developing and developed countries [10]. Similarly, E. coli is a non-spore-forming bacterium and the major cause of foodborne diseases in Gram-negative bacteria. Disease-causing strains of E. coli can infect the stomach, leading to serious abdominal symptoms [11]. Previous studies have primarily concentrated on spore-forming microorganisms, thereby overlooking non-spore-forming ones such as E. coli and S. aureus. Based on their contribution to foodborne illnesses, it is important to develop a cost-effective user-friendly approach to slow their rapid proliferation in food products.

Organic acids have been used as antimicrobial agents to inhibit foodborne pathogenic bacterial growth in chicken meats during processing [12]. Due to the potential resistance development by microorganisms, there are needs of drug alternatives that can efficiently kill resistant bacteria and enhance preservation [13]. Antimicrobial peptides (AMP) are produced by living organisms and include critical functions in protecting hosts against infections [14,15]. Likelihood of microbes exhibiting resistance to AMP is exceedingly low because of their wide range of mechanisms of action. Multiple studies have emphasized potential of AMP as a viable option for preventing meat spoilage and foodborne diseases [16-19]. In a previous investigation by the current authors, recombinant AGAAN (rAGAAN) effectively was cloned, expressed and analytically characterized [20]. Technically, AGAAN is a novel antimi-crobial peptide with a cationic α-helical structure from the skin secretions of the blue-sided frogs. The rAGAAN is stable at various temperatures and pH and destroys a wide range of bacteria [20]. A hemolytic assay has shown that the peptide is relatively non-toxic to mammalian red blood cells (RBCs). Combination of these characteristics with its rapid killing kinetics demonstrates that rAGAAN includes the potential as an effective food preservative against foodborne pathogens. Nevertheless, major issues include exorbitant production expenses, labor-intensive procedures and potential toxicity of using high concentrations of peptides.

Combining two or more AMPs may boost antimicrobial activity at lower doses [21]. The present study assessed pairwise combinations of the rAGAAN with formic and acetic acids against E. coli and S. aureus. Selection of these two organic acids was based on their high effectiveness against the highlighted bacterial strains. In addition, FAO/WHO Expert Committee on Food Additives has classified acetic and formic acids as generally regarded as safe. The former chemical was assigned to an unrestricted group acceptance daily intake (ADI), while the latter was assigned to an ADI range of 0–3 mg.kg^-1 [22]. Combination of rAGAAN and these organic acids could decrease the concentration while preserving their potentially bactericidal activity. Differences in their mechanisms of action necess-itate assessment of synergy in membrane permeation and kinetics of inactivation. This study could provide an additional option for poultry industries to protect chicken meats from pathogens.

1. Materials and Methods

2.1 Bacterial strains

Department of Microbiology at King Mongkut's University of Technology Thonburi in Bangkok, Thailand, supplied the foodborne pathogenic strains of E. coli ATCC 8739 and S. aureus ATCC 6538.

2.2 Recombinant AGAAN peptide expression and purification

The rAGAAN was produced based on the method of Ajingi et al., [20]. Briefly, the recombinant plasmid (pET-AGAAN) was transformed into E. coli BL21 (DE 3) competent cells. A colony of the competent cells with recombinant plasmids was inoculated into Luria-Bertani (LB) broth supplemented with chloramphenicol and ampicillin and grown at 37 °C and 200 rpm overnight. Then, 1% v v^-1 from the overnight culture was introduced into a fresh 1-l LB broth supplemented with chloramphenicol and ampicillin as well as 1% w v^-1 glucose. Culture was grown to an optical density (OD 600 nm) range of 0.4–0.6 at 37 °C and 200 rpm. Then, rAGAAN was expressed through induction with isopropyl β-D-1-thiogalactopyranoside at a concentration of 500 mM. Culture was grown at 16 °C for 18 h at 150 rpm. Cells were collected through centrifugation at 6,120× g for 30 min at 4 °C. Then, cells were suspended in 10 ml of buffer solution (10 mM Tris-HCl, 1 M NaCl; pH 8.0). These were subjected to sonication at an amplitude of 60% for 2 min, repeated for five cycles to induce cell disruption. Supernatant was purified after sonication and centrifugation at 6,120× g for 25 min at 4 ℃ using HisTrap FF column linked to the FPLC system. The column was pre-equilibrated with binding buffer (10 mM Tris-HCl, 1 M NaCl; pH 8.0). Elution of the bound peptide was carried out using buffer B (10 mM Tris-HCl, 1 M NaCl, 250 mM imid-azole; pH 8.0). Then, dialysis was carried out overnight at 4 °C using 50 mM Tris-HCl solution. Then, peptide was concentrated using 3-kDa centricon centrifugal filter tubes (Amicon, Germany). Concentration of the rAGAAN was measured using Bradford protein assay and its purity was assessed using 16% tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (tricine-SDS-PAGE).

2.3 rAGAAN and organic acid preparation

The rAGAAN was formulated in milligrams per milliliter (mg.ml^-1). It was dissolved in 1× phosphate-buffered saline (PBS), whereas the organic acids were formulated in percentages (% v v^-1) by dissolving in distilled water (DW).

2.4 Culture preparation

A volume of 20 μl of microbial stock, previously stored at -80 ◦C, were plated on LB agar. The resulting culture was incubated at 37 ^oC for 18 h. Then, subculture process was carried out for each strain under identical conditions to preserve integrity and purity of the cells. On the next day, a suspension was generated by transferring isolated colonies into sterilized 10-ml LB media. The bacterial strains were cultured until they reached an OD of 10⁸ cfu.ml^-1. This measurement was achieved at 600 nm using spectrophot-ometer (U-2900UV/VIS Hitachi Tokyo, Japan). Concen-tration was modified to 10⁵ cfu.ml^-1 using sterile LB broth.

2.5 Minimum inhibitory concentration assessment

Briefly, 50 μl of the inoculated sample were administered into each well of the 96-well plates. Then, aliquots of 50 μl were dispensed into the wells, containing rAGAAN and organic acids at various concentrations. The 96-well plates with the lids closed were incubated at 37 °C for 18 h. Results were analyzed at 600 nm using microplate reader (BioTek, synergy H1, Winooski, USA). Control contained 100 μl of the bacterial inoculum. The MIC values included the lowest concentrations of the antimicrobial agents that cause bacterial growth inhibition.

2.6 Synergistic effects of rAGAAN with acetic and formic acids

Combination effects of rAGAAN with organic acids against the bacterial strains were assessed using checkerboard method. Briefly, 18-h cultures in LB broth were used to inoculate fresh LB broth to achieve a cell density of approximately 10⁵ cfu.ml^-1. Generally, 50 μl of the inoculated sample were added into 96-well microplates. Then, rAGAAN and organic acids were transferred into the 96-well microplates with increasing concentrations arran-ged in columns and rows, respectively. The organic acids were mixed with rAGAAN separately to assess their combinatorial effects on pathogenic bacteria. The purpose was to decrease the effective concentration of rAGAAN while preserving its antimicrobial activity. Assessment of the synergistic interactions involved the summation of the fractional inhibitory concentration indices (FICI) as Eq. 1 [23].

                                                                                                 Eq. 1

where, FICI ≤ 0.5 indicated synergistic relationships between the rAGAAN and organic acids that increased the antimicrobial activity, FICI > 0.5–4.0 was indifferent and FICI > 4.0 was antagonistic.

2.7 Kinetics of inactivation

The OD of bacterial strain was measured to assess the rate of inactivation when treated with rAGAAN, acetic acids or their combination. Bacterial culture, diluted in LB broth to a concentration of approximately 10⁵ cfu.ml^-1, was added to 96 well plates. The rAGAAN and acetic acid were added at their minimum inhibitory concentration (MIC) levels, individually and in combination with their fractional inhibitory concentration (FICI) at 1×, 2× and 3× to separate wells. The 96-well plate was incubated at 37 °C. The procedure entailed monitoring the rate of inactivation for various bacterial strains by measuring the OD at consistent intervals of 1 h for 5 h. The OD was measured using spectrophotometer set at 600 nm and microplate reader (BioTek, synergy H1, Winooski, USA).

2.8 β-Galactosidase assay

The β-galactosidase assay was carried out to assess effects of the rAGAAN and acetic acid or their combination on membranes of the bacteria. First, E. coli was inoculated into lactose broth and incubated at 37 °C for 18 h to stimulate β-galactosidase production. The bacterial cells were centrifuged and the pellet was washed thrice with 1× PBS. Then, the bacterial concentration was modified to roughly 10⁵ cfu.ml^-1 in 1× PBS solution. Moreover, 50 μl of purified rAGAAN, acetic acid and their combination at 1× FICI, 2× FICI and 3× FICI were added into wells of a microplate containing 50 μl of E. coli cell suspension. A volume of 30 μl of O-nitrophenyl-β-D-galactoside (ONPG) were added into every well of the microplate. The microplate was incubated at 37 °C and activity was assessed by measuring the spectrophotometric absorbance at 405 nm and various time intervals.

2.9 Hemolysis assay

The RBC lytic assay was carried out based on a procedure by Taniguchi et al. [24] with minor adjustments. The RBCs were washed thrice in 1× PBS and centrifuged at 14,530× g for 10 min. Pellet was dissolved in 1× PBS to achieve a concentration of 4%. Generally, 500 μl of blood were mixed with 500 μl of rAGAAN and acetic acid, indivi-dually and in various combinations (1×FICI, 2×FICI, and 3×FICI). The positive control included a solution containing 0.1% TritonX-100, while the negative control included a solution containing 1× PBS. Solution was incubited in microtubes at 37°C for 1 h and centrifugation was carried out at 14,530× g for 5 min. Then, 100 μl of the supernatant were extracted from each microtube and transferred to each well of 96-well plate. Assessment of hemoglobin release was carried out by measuring the absorbance at 540 nm.

2.10 rAGAAN-acetic acid against chicken meat spoilage

Antimicrobial efficacy of the rAGAAN and acetic acid combination was assessed using a methodology described by Ajingi et al. [25], with minor adjustments. In brief, fresh chicken meat was purchased from a local market and immediately transferred to the laboratory. Meat was divided into approximately 10-g specimens and washed thoroughly. Specimens were transferred into a laminar flow hood and 100 µl of 10⁵ cfu of E. coli were divided to five separate locations. Sample was set for 1 h to promote appropriate attachment of the bacterial strains. Then, meat sample was submerged into 200-ml solution of rAGAAN/acetic acid for 1 h. Furthermore, sample was extracted, transferred into a plastic bag and incubated at 37 ^oC for 3 d. The chicken meat sample was transferred into a plastic bag with solution consisting of 0.1% peptone water. Sample was mechanically pulverized using stomacher to enhance liberation of the bacterial cells. Following the process of serial dilution, a 100 µl of sample were transferred onto an LB-agar plate. Number of colonies on the plate was counted at intervals of 0, 1, 2 and 3 d. Control group was administered with DW.

2.11 Statistical analysis

Results were present as mean ±SD (standard deviation) of three replicates. Statistical distinction was assessed using one-way analysis of variance (ANOVA) with Duncan’s multiple-range test. Differences with p < 0.05 were regarded as statistically significant.

1. Results and Discussion

3.1 Minimum inhibitory concentration

The MICs of rAGAAN and organic acids against S. aureus and E. coli are present in Table 1. The MIC of rAGAAN against S. aureus and E. coli was assessed as 0.15 mg.ml^-1. The organic acids inhibited proliferation of the pathogenic bacteria at various concentrations expressed as proportions (%).

Acetic acid demonstrated inhibitory effects on the growth of S. aureus at 0.2% v v^-1 and on the growth of pathogenic E. coli at 0.25% v v^-1. Formic acid inhibited growth of S. aureus at 0.25% v v^-1 and growth of E. coli at 0.2% v v^-1. The findings for acetic acid were similar to those against eleven mastitis pathogens in dairy cows with MIC values ranging of 0.125–0.25% v v^-1 [26]. Similarly, Fraise et al. [27] reported antimicrobial activity of acetic acid against Pseudomonas aeruginosa and S. aureus at 0.166 and 0.312% v v^-1, respectively. Manuel et al. [28] detected that formic acid at a concentration of 0.06% v v^-1 exhibited antimicrobial effects against E. coli. Variations in their effectiveness against the microorganisms might be attributed to their chemical compositions. Methyl group (CH3) in acetic acid donated electron density to O-H bond, resulting in increased difficulties in removing the hydrogen atom. Consequently, acetic acid was weaker than the formic acid. Weak acids included a higher ability to pass through bacterial membranes, compared to strong acids due to the balances between their ionized and non-ionized states. The non-ionized form could easily diffuse through hydrophobic membranes. As a result, they provided proton gradients needed for ATP synthesis to collapse. This occurred because free anions such as acetate in this situation combined with periplasmic protons that were pumped out by the electron transport chain. Then, anions transported the protons back across the membrane without F1Fo ATP synthase [29].

3.2 Synergistic effects of rAGAAN with organic acids

The inhibitory concentration index (FICI), demons-trating combined effects of rAGAAN and organic acids, is present in Table 2. The compound rAGAAN demonstrated synergistic effects against S. aureus and E. coli when combined with organic acids. Results showed that the synergistic effects were strongest when using acetic acid for the two bacterial strains, compared to when using formic acid. The FICI values for the combination of rAGAAN with acetic acid were assessed as 0.375 (p < 0.5) for S. aureus and E. coli. The FICI values for the combination of rAGAAN with formic acid were assessed as 0.375 for S. aureus and 0.5 for E. coli.

Results indicated that sub-MICs of the antimicrobials were needed to effectively terminate the bacterial growth. Combination of rAGAAN and acetic acid resulted in a 25% decrease in the concentration of each antimicrobial, compared to their MICs. Synergism can occur when two various antibacterial agents, each with non-overlapping mechanisms of action, are combined with each other [30]. Therefore, the authors suggest that the antimicrobial effects could be strengthened using synergistic effects of combined organic acid with rAGAAN. While the precise process; by which, combination of rAGAAN with organic acid created synergistic effects is still unknown, studies have demonstrated that the cell membrane of bacteria is a shared target for the antibacterial effects of various antimicrobial peptides. Additionally, these peptides include an affinity for bacterial cellular components, including DNA [31]. In contrast, it is suggested that organic acids can delay absorption of nutrients and disrupt flow of electrons, leading to decreases in ATP production [32]. This various mechanism of action enables swift eradication of bacteria.

3.3 Kinetics of inactivation

Acetic acid was chosen for the study because it included stronger antimicrobial effects than that formic acid with rAGAAN did. Growth inhibition kinetics of rAGAAN, acetic acid and their combination on the logarithmic phase of the pathogenic bacteria are illustrated in Figure 1. When the peptide rAGAAN was mixed with acetic acid at the FIC, there was no noticeable alteration in OD for either of the bacterial strains during 5 h. This indicated that the bacterial growth was entirely suppressed. The combination demonstrated significant inhibitory effects, greater than that of the individual antimicrobial agent and control group. The combination exhibited the capacity to inhibit proliferation of S. aureus ATCC 6538 and E. coli ATCC 8739 at various concentrations within a few hours of exposure. Upon analyzing each treatment individually, it became clear that progressive decreases occured in OD measurements as time progressed. Nevertheless, use of rAGAAN with acetic acid led to further pronounced decreases in the turbidity level of the culture.

3.4 β-Galactosidase assay

To clarify the mechanism; by which, the combination acted, membrane permeability assay was carried out. This experiment used E. coli that was cultured in media containing lactose broth, which stimulated the synthesis of β-galactosidase. The β-galactosidase is an endogenous enzyme synthesized by the lac operon in bacteria. Release of this enzyme depends on disruption of the cell membrane. Release of the β-galactosidase enzyme from the disrupted cytoplasmic membrane was detected within 10 min of incubation with rAGAAN alone. In addition, cell membrane was destabilized by a combination of rAGAAN with acetic acid at 1× FICI, 2× FICI and 3× FICI, as shown in Figure 2.

Findings showed that the presence of rAGAAN, independently and in combination with acetic acid, could result in permeability of the cell membrane of E. coli. How-ever, acetic acid alone did not demonstrate effects on perme-ability of the membrane. Increasing OD measurements over time were directly linked to the rate of O-nitrophenol prod-uction from the breakdown of ONPG. The current results were similar to those of Yuan et al. [33], who observed increases in OD due to the degradation of ONPG when Larimichthys crocea myosin heavy chain protein-derived peptide was combined with low-intensity ultra-sound. Cell membrane disruption might lead to releases of intracellular components [34].

3.5 Hemolysis assay

Potential cytotoxicity of the chemical combination on human RBCs was assessed. Results of the hemolytic assay are present in Figure 3. The experimental group supernatant included clarity and transparency, which sharply contrasted to that the positive control group supernatant did. Further-more, no substantial fluctuation was recorded in absorbance of the experimental groups. This demonstrated that combin-ation of acetic acid and rAGAAN did not include any adverse effects on RBCs. However, absorbance of acetic acid alone resulted in approximately 50% hemolysis of the RBCs presented at high concentrations. Results suggested that the two antibacterial agents could effectively be combined as harmless food additives to combat pathogens.

3.6 rAGAAN-acetic acid against chicken meat spoilage

Results of the experiment are shown in Table 3 and Figure 4. Use of rAGAAN and acetic acid, either separately or in combination, led to significant suppressions of the E. coli growth. Inhibition was observed at various durations of exposure, in contrast to the control group (Table 3). At the beginning of the experiment (Day 0) after exposure to DW for 1 h, the control group showed decreases in the cell population to a value of 6.06 log 10 cfu.g^-1. Significant decreases (p = 0.05) were observed in the bacterial count after individual treatment with rAGAAN and acetic acid (4.87 and 5.01 cfu.g^-1, respectively). Similarly, when the combination therapy was administered at concentrations of 1×, 2× and 3×, the resulting bacterial counts were 4.85, 4.82 and 4.81 cfu.g^-1, respectively. Significant differences in decreases were reported when comparing the control group to the groups that received rAGAAN treatment alone and the groups that received combination treatments at a three-fold fractional inhibitory concentration index as the duration of treatment increased. On Day 1 of the experiment, statistically significant decreases were seen in values of the microorganisms. More precisely, rAGAAN alone and its combination resulted in decreases of 1.39 and 1.40 log 10 cfu.g^-1, respectively.

On Day 2, the 3× FICI combination group showed statistically significant decreases of 1.53 log 10 cfu.g^-1, compared to the control group (p = 0.05). Furthermore, significant decreases of 1.63 log 10 cfu.g^-1 were reported on Day 3, compared to the control group. Results of this study demonstrated that use of rAGAAN and acetic acid included combined antibacterial effects on E. coli in meat samples stored at 37 °C for 3 d. The synergistic effect was detected as superior to the antibacterial effects of rAGAAN when used separately. Results supported changes in the color of the meat. The transformation is documented through the alteration in color of the meat (Figure 4). The control group demonstrated a significant color change and included an unpleasant smell. This might be attributed to the prolif-eration of microbial organisms. Katiyo et al. [35] reported strong correlations between the odor and growth of microorganisms in chicken legs. The putrid smell of spoiled meat might be due to the existence of various compounds such as sulfur compounds, carbonyls, ketones, diamines and alcohols [36].

1. Conclusion

This study provided evidence on the synergistic effects of combining rAGAAN antimicrobial peptides with organic acids to inhibit growth of two prevalent foodborne pathogens of S. aureus and E. coli. Significantly, this integr-ated approach created inhibitory effects at decreased con-centrations, compared to separate uses of these substances. The study revealed that rAGAAN included the highest beneficial synergy when combined with acetic acid. Simultaneous administration of rAGAAN and acetic acid caused disruption of the bacterial cell membrane without causing detectable harms to mammalian RBCs. Further-more, implementation of multiple treatments led to decre-ases in the presence of microorganisms in chicken meats after 3 d, compared to the group with no treatments. The study results can be used as secure economically feasible alternatives for the preservation of chicken meats. While the current study assessed two pathogens, further studies are necessary to investigate effects of the chemical combi-nations on a wider range of microorganisms as well as assessing precise mechanisms of the synergistic intera-ctions.

1. Acknowledgements

This study was supported by research funds from Petchra Pra Jom Klao Research Scholarship (contract no. 74/2563) and Faculty of Science, King Mongkut’s University of Technology Thonburi.

1. Conflict of Interest

The authors declare no conflict of interest.

1. Authors Contributions

Investigation, writing-original draft, data curation and formal analysis, N.U.J.; formal analysis, validation, Y.S.A.; Data curation, formal analysis, N.R.; project administration and supervision, T.R.; methodology, supervision, W.S.; project administration and supervision, P.P.; supervision, conceptualization, funding acquisition, resources, methodology, N.J.

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