Abstract
Antimicrobial activities of aminoreductone (AR), a product formed in the initial stages of the Maillard reaction, were investigated using eight clinical isolates of antibiotic-susceptible/resistant pathogenic bacteria: four Pseudomonas aeruginosa, one multi-drug-resistant Pseudomonas aeruginosa (MDRP), one Escherichia coli, one methicillin-susceptible Staphylococcus aureus, and one methicillin-resistant Staphylococcus aureus (MRSA). AR showed advanced growth inhibition effects compared to antibiotics (amikacin, ciprofloxacin, imipenem and levofloxacin) frequently used for the treatment of infectious diseases, and worked synergistically with these antibiotics. The minimum inhibitory concentrations (MIC) of AR ranged from 13 to 20 mM. The bactericidal activity of AR was dose and time dependent. The minimum bactericidal concentration (MBC) was lower for Gram-negative bacteria (32 to 40 mM). These findings suggest that AR, a naturally formed antimicrobial agent present in thermally processed foods, has promising potential for health promotion and for use in medical practices.
Introduction
Since their discovery, antibiotics have provided the main basis for the treatment of infectious diseases caused by a variety of microorganisms in medical practices (Singh et al., 2010) . However, overuse and/or abuse of antibiotics have become the main factor in the emergence and dissemination of multi-drug-resistant strains of bacteria (Singh et al., 2010) . Multi-drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug-resistant Pseudomonas aeruginosa (MDRP), are widely distributed in hospitals and are increasingly being isolated from community-acquired infections (Rao and Wong, 2003; Khan et al., 2004) . Currently, antibiotic resistance in pathogenic bacteria is a significant clinical problem and a serious public health concern (Manikandan et al., 2011) . Thus, the identification of new antimicrobial agents is a top research and development priority among scientists and pharmaceutical companies (Alekshun and Levy, 2007) .
Introduction
The Maillard reaction, a heat-treatment-induced chemical reaction between amino and carbonyl groups, is important in food processing because it strongly affects product quality and acceptance (van Boekel, 1998) . Several authors have reported beneficial effects of advanced Maillard reaction products (MRP) such as melanoidins, including antioxidative (Hiramoto et al., 2004; Echavarría et al., 2012) , antimicrobial (Einarsson et al., 1983; Rufián-Henares and Morales, 2007a , 2007b , antihypertensive (Rufián-Henares and Morales, 2007b) , antimutagenic, and anticarcinogenic properties (Gu et al., 2010) . Concerning antimicrobial activity, several reports highlighted the in vivo and in vitro effects of monohydrate (262 mmol L −1 ) and butylamine (1.16 mol L −1 ) were dissolved in 1.28 mol L −1 phosphate buffer (pH 7.0).
Introduction
The sample solution (10 mL) was heated at 100℃ for 15 min, and immediately cooled on ice. The heated sample solution was extracted three times with a double volume of ethyl acetate, and the ethyl acetate layer was evaporated to dryness under reduced pressure. The residue was dissolved in 10 mL of 20% methanol and filtered through a Sep-Pak Plus C18 cartridge (Waters Corporation, Milford, MA) (activated by 5 mL of ethanol and equilibrated using Milli-Q water) to remove brown components (melanoidins). The clear eluate was evaporated again and freeze-dried under reduced pressure to collect the purified AR. In a previous study, Shimamura et al. (2004) reported the 13 C-and 1 H-NMR data on this extracted product, and their signals were assigned to AR (1-(butylamino)-1,2-dehydro-1,4-dideoxy-3-hexulose). The concentration of AR was calculated with the extinction coefficient of AR (17.98 M −1 cm −1 ) at 320 nm (Trang et al., 2008) . Brown, high-molecular-weight polymerized products are formed in the final stages of the Maillard reaction. These were named melanoidins by the French chemist Louis Camille Maillard (van Boekel, 1998) . Because melanoidins showed antimicrobial activity against several pathogenic strains, as described above (Lanciotti et al., 1999 , Rufián-Henares and Morales, 2007a , 2007b Rufián-Henares and Cueva, 2009 ), AR-derived MRP, which is a mixture of advanced MRP mainly containing melanoidins, was also used in this study. AR-derived MRP was prepared as follows: the purified AR solution was kept at room temperature for at least 3 weeks until the remaining amount of AR was less than 0.5%, as confirmed by the XTT assay or by estimating the absorbance at 320 nm (Trang et al., 2009) .
Introduction
Bacterial strains and culture conditions The eight antibiotic-susceptible and -resistant pathogenic isolates, 4 PA (P. aeruginosa), 1 MDRP (multi-drug-resistant P. aeruginosa), 1 EC BL 21 (E. coli), 1 MSSA (methicillin-susceptible S. aureus) and 1 MRSA (methicillin-resistant S. aureus), were obtained from patients at Kochi Medical School Hospital (Kochi, Japan). Two reference strains, S. aureus ATCC 25923 (SA 25923) and E. coli ATCC 25922 (EC 25922), obtained from the American Type Culture Collection were used as control strains to investigate the interaction between AR and antibiotics. All strains were grown on MHB agar plates supplemented with 1.4% agar and incubated at 37℃ for 24 h under aerobic conditions.
Introduction
Disc diffusion susceptibility methods The growth inhibition of pathogenic strains was assessed using the filter paper disc diffusion method on MHB agar plates, incubated at 37℃ under aerobic conditions (Nakhaei et al., 2008) . Purified AR melanoidins against Bacillus stearothermophilus, a highly thermo-resistant microorganism involved in the degradation of food (Rufián-Henares and Morales, 2007b; Lanciott et al., 1999) , and pathogenic and spoilage bacteria frequently found in food, such as Escherichia coli (Rufián-Henares and Cueva, 2009 ), S. aureus (Einarsson et al., 1983; Rufián-Henares and Morales, 2008a; Rufián-Henares and Cueva, 2009) Pischetsrieder et al. (1998) in a heated solution of lactose and N α -acetyllysine (Pischetsrieder et al., 1998a) . Shimamura et al. (2011) reported the formation and presence of AR in milk using a combination of 2,4-dinitrophenylhydrazine (DNP) and Cu 2+ (Shimamura et al., 2011) . Because AR can be detected after only a short period of heating, it is an important indicator for estimating the extent of the Maillard reaction and heat treatment of food (Shimamura et al., 2004) . Elucidation of the role and characteristics of AR are, therefore, of great interest to food scientists. To date, antioxidant activity (Pischetsrieder et al., 1998b) , protective effects on photo-degradation of riboflavin (Trang et al., 2008) , and antimicrobial activity against Helicobacter pylori, a Gramnegative bacilli (Trang et al., 2009) , have been reported for AR (1-(butylamino)-1,2-dehydro-1,4-dideoxy-3-hexulose), derived from the Maillard reaction of lactose and butylamine. In the search for alternatives to antibiotics, and to further describe the functional properties of AR, this study investigated the effects of AR against pathogenic bacteria, including P. aeruginosa (PA), multi-drug-resistant P. aeruginosa (MDRP), E. coli (EC), methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).
Materials and Methods
Reagents Mueller-Hinton broth (MHB) and commercially available standard discs (ϕ = 6 mm) such as amikacin (AN; 30 μg disc −1 ), ciprofloxacin (CIP; 5 μg disc-1), imipenem (IPM; 10 μg disc −1
Materials and Methods
) and levofloxacin (LVX; 5 μg disc −1 ) were obtained from Becton, Dickinson and Company (Franklin Lakes, New Jersey). Lactose monohydrate was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). n-Butylamine and agar were obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were of the highest commercial grade available. Milli-Q water or sterilized water was used in all procedures.
Materials and Methods
Preparation of AR and its degradation product Purified AR was prepared according to previous reports (Pischetsrieder et al., 1998; Shimamura et al., 2004) . Briefly, lactose incubated under aerobic conditions with shaking (Bio shaker BR-40LF, Taitec Co., Ltd., Saitama, Japan) at 37℃ for 7 h. At 1, 3, 5 and 7 h after incubation, each 10 µL of the suspension was serially diluted 10-fold, inoculated onto the MHB agar plates and cultured for 24 h under aerobic conditions to determine cell viability. The ability of AR to kill pathogenic strains was evaluated by CFU counts in comparison with controls. All examinations were performed in triplicate.
Materials and Methods
Cluster analysis Based on the results of the susceptibility tests with antibiotics and AR, all eight isolates were subjected to cluster analysis (Townend, 2002) .
Materials and Methods
Statistical analysis All experimental data were analyzed using an analysis of variance (ANOVA) and significant differences (P < 0.05) among means, from triplicate values, were determined using Duncan's multiple range tests with SPSS (v.12.0 for windows, SPSS Inc, Chicago, IL).
Results

Inhibitory effects of AR against pathogenic bacteria
The inhibitory effects of AR against eight pathogenic isolates and two reference strains were examined using the standard disc diffusion and the agar dilution methods. Table 1 shows the inhibition zone by AR (2.5 mg) for each isolate. Inhibition zones ranged from 9.5 mm (MDRP) to 28.3 mm (SA 25923) in diameter, suggesting that all isolates were sensitive to AR at the tested concentration.
Inhibitory effects of AR against pathogenic bacteria
Previously, it was reported that the addition of Cu 2+ could drastically reduce the concentration of AR because a labile reductone structure in AR could be readily oxidized (Shimamura et al., 2004) . In the presence of Cu
2+
, a considerable decrease in inhibition zone was observed for all isolates due to decreased concentrations of AR. The disc containing 5 μg of Cu 2+ alone showed no inhibitory effect, indicating that AR per se possessed the potential ability to inhibit the growth of all pathogenic bacteria. AR has a labile reductone structure and can easily change into intermediate and advanced MRP, including melanoidins, possessing antimicrobial activities (Einarsson et al., 1983; Rufián-Henares and Morales, 2008b; Rufián-Henares and Cueva, 2009 ). Next, AR-derived MRP, including melanoidins, was utilized in the growth inhibition assay at a concentration similar to each MIC value of AR. However, no inhibitory effect was observed, showing that the AR-derived MRP, containing melanoidins, had no effect against any of the pathogenic strains under our experimental conditions (data not shown).
2+
The inhibitory effects of AR against all isolates were confirmed by an agar dilution method at concentrations lower than 20 mmol L −1 (Table 1) . MIC values range from 13 mmol L −1 (MSSA) to 20 mmol L −1 (MDRP and PA4). The was diluted in Milli-Q water and dropped onto the disc. Sterilized standard discs (ϕ = 6 mm) containing 2.5 mg of AR were placed on the MHB agar plates previously spread with 0.1 mL of bacterial suspension (OD 600 = 0.1) in MHB liquid medium. The plates were incubated at 37℃ for 24 h under aerobic conditions. The inhibition zones were measured and recorded in millimeters, and the average diameter of at least two repetitions was calculated. The entire diameter of the inhibition zone, including the diameter of the disc, was measured. Four standard commercially available antibiotic discs such as amikacin (AN: 30 μg disc ) were also used in this assay. An inhibition circle of more than 6 mm was defined as anti-bacterial activity.
2+
Previously, Shimamura et al. (2004) observed that the addition of Cu 2+ , a strong oxidizing agent, drastically decreased AR content in the lactose-butylamine model solution. To confirm the contribution of AR to the inhibitory activities in this study, Cu 2+ (5 µg) was added to the disc containing AR (2.5 mg) and changes in the inhibition zone were observed.
Determination of minimum inhibitory concentrations (MIC)
MIC of AR against pathogenic strains was determined using a previously described agar dilution method (Dore et al., 1999; Nakhaei et al., 2008) . Purified AR was diluted in Milli-Q water. Aliquots (750 µL) of AR solution at given concentrations were added separately to each dish containing 14.25 mL of not-yet solidified MHB agar. The final concentrations of AR in the agar plates ranged from 0 to 30 mmol L −1 . Subsequently, each 10 µL of pathogenic strain suspension (OD 600 = 0.1) was serially diluted 10-fold and inoculated onto the surface of the AR-supplemented agar plates, then incubated at 37℃ for 48 h under aerobic conditions. Sterilized water was used as a control for all experiments. The number of colony forming units (CFU) was determined as a measure of bacterial viability (Takeuchi et al., 2006) . MIC was defined as the lowest AR concentration to inhibit 1 × 10 4−5 CFU mL −1 compared to controls. In addition, in this study, AR-derived MRP was also used and compared with AR. All tests were performed in at least triplicate. Killing assay To determine the bactericidal activity of AR against pathogenic bacteria, killing experiments were performed in the presence of 2, 5 or 10 × MIC of AR, according to the previously described method (Dore et al., 1999) . After 24 h of culture in MHB liquid medium, corresponding to the late exponential phase, the bacteria were harvested, washed with MHB liquid medium and then centrifuged at 8,000 g for 1 min (KUBOTA 1120, Kubota Corp., Tokyo, Japan) to remove the supernatant. In 1.5-mL centrifuge tubes, 0.4 mL of the bacterial suspension (10 9 CFU ml −1 ) in fresh MHB liquid medium with AR or without (controls) were including MDRP, followed by E. coli at a concentration of 2 × MIC (36 mmol L −1 ) of AR after 3 h of incubation. The biological behavior of the two S. aureus isolates did not differ significantly at any concentration of AR during the first 5 h of incubation. However, the decrease in the concentration of MRSA appeared at 7 h, seemingly in a time-dependent manner. MSSA appears to be the most resistant strain to AR; its concentration decreased to 10 6 CFU mL −1 at 7 h with 10 ×
MIC of AR.
Verification of the antimicrobial activity of AR in combination with antibiotics Drug interactions can be beneficial (synergistic or additive interaction) or deleterious (antagonistic or toxic outcome) (Adwan and Mhanna, 2008) . The disc diffusion assay utilizing antibiotic and AR discs and the discs containing the mixtures demonstrated synergy, but no susceptibility of all isolates to widely used antibiotics (LVX, CIP, IPM and AN) compared with AR is listed in Table 1 . Interestingly, all isolates, including MDRP, were susceptible to AR (2.5 mg). Cluster analysis completely separated the two S. aureus isolates from the other strains (data not shown).
MIC of AR.
Killing ability of AR against pathogenic bacteria AR inhibited the bacterial growth of all isolates tested, except for MSSA. The in vitro activities of AR were further evaluated in killing assays with 2, 5 or 10 × MIC of AR to determine the minimum bactericidal concentrations (MBC) ( Table 2) . The bactericidal effects, a reduction of more than 10 4−5 CFU mL −1 of culturable cells, were mainly observed after 7 h of incubation for all isolates except MSSA (Figure 1) . However, the effects were individual; the most potent killing activity was dose-dependently exhibited in P. aeruginosa isolates, 1 kind of antibiotic (IPM). AR is a product formed in food during heat treatment and is not a medicine, which may possess side effects. Thus, similar to other active products found in foods or herbs, a higher amount of AR is needed to exert antimicrobial activity compared with antibiotics. The amount of AR obtained depends on the food source. For example, 25 mg of AR can be gained from 200 mL of UHT milk (Trang et al., 2009) . These amounts are achievable for daily consumption. Furthermore, we confirmed that AR exhibited growth inhibition, even against antibiotic-resistant strains. These results revealed that AR showed advanced growth inhibition antagonistic effects, even in drug-resistant isolates, indicating that AR does not interfere with the activity of antibiotics (Figure 2 ).
Discussion
Pathogenic bacteria that have developed resistance to multiple antibiotics, particularly MRSA and MDRP, are of grave concern to immunocompromised patients, as they are responsible for nosocomial infections. In this study, PA and EC BL 21 showed resistance to three kinds of antibiotics (LVX, CIP, IPM) and MRSA demonstrated resistance to (Darville, 1999) or AN (Carrier et al., 1997) are widely used alone or in combination with other antimicrobial agents for the treatment of several serious infections caused by Gramnegative and -positive bacteria. Combined antibiotic therapy has been shown to delay the emergence of bacterial resistance and may also produce desirable synergistic effects in clinical settings (Adwan and Mhanna, 2008) . The experiments combining antibiotics and AR were carried out by the standard disc diffusion method, which is widely used to study the bioactivity of chemical compounds (Rufián-Henares and Morales, 2008a) . The ability of compounds to diffuse through the nutrient agar medium is a major limitation in the evaluation of the antimicrobial effects of the compounds concerned. The contact area of the two inhibition zones of antibiotic and AR expressed the effects of both AR and antibiotic against bacteria. Synergy has generally been defined as effects at the tested concentration compared to antibiotics. Thus, AR could be used either as an alternative antimicrobial agent or as an adjuvant therapy for infectious diseases. AN (amikacin), an aminoglycoside, inhibits protein synthesis by "irreversibly" binding to the 30S ribosomal subunit, thus preventing the formation of an initiation complex with messenger RNA (Carrier et al., 1997) . Of the four antibiotics used, AN showed the highest inhibitory effect, similar to the effect of AR (except for MDRP), suggesting that AN could be used as a reliable reference during investigation of AR antimicrobial activity. AN has been generally recognized to have some serious adverse effects, such as nephrotoxicity and permanent vestibular and/or auditory ototoxicity (Carrier et al., 1997) ; hence, the use of AR could be an appropriate alternative to AN. The fluoroquinolone antibacterial agents LVX and CIP (Mori et al., 2000) , β-lactam antibiotics (IPM) function of AR might be due to the thickness of the bacterial cell wall, as well as the outer membrane-related components. In other words, AR efficacy might depend on the constitutive elements of bacterial structures. To elucidate this issue, further investigations using improved AR or AR-derived compounds, and identification of the biochemical characteristics of AR, including its molecular structure, are required.
Discussion
To date, the majority of attention has been paid to highmolecular-weight MRP, such as melanoidins, on the topic of antimicrobial activity. It was reported that the growth inhibitory activity and bactericidal activity against E. coli increased depending on the molecular weight of MRP (< 3 kDa, 3-10 kDa and > 10 kDa) (Rufián-Henares and Morales, 2008b) . Einarsson et al. (1983) showed that the growth inhibitory activity of MRP with molecular weight > 1 kDa was higher than that with < 1 kDa molecular weight. However, our study demonstrated for the first time that low-molecular weight MRP such as AR (217 Da) can exhibit bacteriostatic and bactericidal properties. It is noteworthy that AR possesses antimicrobial activity against pathogenic bacteria. AR from lactose and butylamine showed high reducing activity (Shimamura et al., 2004) . Lanciotti et al. (1999) suggested that the inhibitory effect against B. stearothermophilus was influenced by the reduction of the redox potential in the growth medium, as a consequence of the addition of MRP with high reducing properties. Therefore, the effect of AR against pathogenic bacteria might be partially caused by its reducing ability.
Discussion
The killing assay has been suggested to be one of the most reliable methods for determining the susceptibility of microorganisms to antimicrobial compounds and antibiotics (Dore et al., 1999) . The killing effect of AR was not associated with drug susceptibility, but appears to depend on bacterial morphology, such as in Gram-positive cocci and Gramnegative rods. According to Oussou et al. (2008) , the ratio of MBCs to MICs (MBC:MIC < 4) indicated that the effect of AR was bactericidal for all Gram-negative strains tested, while it was bacteriostatic for MRSA (MBC:MIC = 10) (Oussou et al., 2008) . In our study, Gram-positive S. aureus isolates were more resistant to AR than the other Gram-negative isolates, confirming the results of the cluster analysis (Table 2 and Figure 1) . Furthermore, MRSA showed higher sensitivity than MSSA at 10 × MIC, implying that a higher concentration than 10 × MIC of AR may be required to kill MSSA. Such a difference in the effectiveness of AR between Gram-positive and -negative bacteria is an important clue in discovering the molecular function of AR in microorganisms. In addition, the antibacterial functions of AR probably differ from those of the antibiotics used in the present study.
Discussion
Here, we report for the first time that AR inhibits the the increased inhibitory effect of antimicrobials in combination compared with each used alone. As showed in Fig. 2 , an obscure border area was observed at the contact point of the inhibition zones induced by AR and antibiotics (Fig. 2) . In the zoom-in figures (B and C), the bacterial inhibition (arrow) beyond the inhibition circles (white line) was observed as the synergistic effect of antibiotics (LVX, AN) and AR. Furthermore, in drug-resistant isolates, several colonies appeared within the inhibition zone of the discs containing LVX, CIP or IPM. This phenomenon indicated the tested antibiotics are not intended to inhibit those organisms. These drug-resistant strains will reduce the effectiveness of an antibiotic. However, no bacteria were observed in the inhibition zones when AR or the mixtures were used in combination, implying that the combination of antibiotics and AR is useful and may prevent the occurrence of resistant strains. On the other hand, in the disc diffusion method with AR and antibiotics, and the combination of these agents, no bacterial growth (colony) was observed in the area where the two agents overlapped (the inhibition zones, as well as the contact area of the two inhibition zones of AR and antibiotics) (Figure 2 ). These results indicated that no analogical effect was observed with combinations of antibiotics and AR. In other word, AR does not repress the inhibitory effect of antibiotics. In fact, the synergistic effect observed suggested that AR might enhance the inhibition effect of tested antibiotics in the treatment of pathogenic bacteria. These results lead us to consider combinatorial approaches based on foods containing AR for daily health and medical application. The exact mechanism by which Maillard reaction products affect bacterial growth is not yet understood. However, it has been suggested that high-molecular-weight compounds with anionic charge, like melanoidins, could develop antimicrobial activity by binding essential metals, such as iron, copper, and zinc, which are key elements in cell metabolism and are essential for the growth and survival of pathogenic bacteria (Rufián-Henares and Morales, 2008b; Rufián-Henares and Cueva, 2009 ). The antimicrobial activity of the Maillard reaction compounds involved interference with the uptake of serine, glucose, and oxygen, thereby inhibiting the sugar catabolizing enzymes of microorganisms by causing irreversible changes in both the inner and outer cell membranes, followed by inhibition of nutrient transport (Lanciotti et al., 1999) . It is thought that the functional activity of melanoidins against Gram-negative bacteria involves membrane damage, based on cell integrity and outer-inner membrane permeability (Rufián-Henares and Morales, 2008b) . Our results demonstrated that AR was more active against Gramnegative (P. aeruginosa and E. coli) than Gram-positive strains (S. aureus), suggesting that the effective antimicrobial growth and viability of pathogenic bacteria, irrespective of their drug susceptibility. In addition, AR was shown to be bactericidal and its effect was enhanced in combination with antibiotics. The antimicrobial effect of AR raises the possibility that foods containing AR could be valuable sources of antimicrobial compounds. Thus, investigating the mechanisms by which AR functions against pathogenic bacteria should aid in understanding its potentially extensive applications in health and medical fields. Foods containing AR, such as milk and dairy products, may be a promising and effective source of AR as an adjuvant. Overall, this study provides useful information to identify the technological conditions that favor the formation of Maillard reaction products, such as AR or melanoidins, as functional ingredients in food.

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