L‑histidine augments the oxidative damage against Gram‑negative bacteria by hydrogen peroxide

  • Authors:
    • Tamiko Nagao
    • Haruyuki Nakayama‑Imaohji
    • Miad Elahi
    • Ayano Tada
    • Emika Toyonaga
    • Hisashi Yamasaki
    • Katsuichiro Okazaki
    • Hirokazu Miyoshi
    • Koichiro Tsuchiya
    • Tomomi Kuwahara
  • View Affiliations

  • Published online on: February 7, 2018     https://doi.org/10.3892/ijmm.2018.3473
  • Pages: 2847-2854
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Abstract

Excessive damage to DNA and lipid membranes by reactive oxygen species reduces the viability of bacteria. In the present study, the proliferation of recA‑deficient Escherichia coli strains was revealed to be inhibited by 1% L‑histidine under aerobic conditions. This inhibition of proliferation was not observed under anaerobic conditions, indicating that L‑histidine enhances oxidative DNA damage to E. coli cells. Reverse transcription‑quantitative polymerase chain reaction analysis demonstrated that the expression of recA in E. coli MG1655 increased ~7‑fold following treatment with 10 mM hydrogen peroxide (H2O2) plus 1% L‑histidine, compared with that following exposure to H2O2 alone. L‑histidine increased the genomic fragmentation of E. coli MG1655 following exposure to H2O2. In addition, L‑histidine increased the generation of intracellular hydroxyl radicals in the presence of H2O2 in E. coli cells. Next, our group investigated the disinfection properties of the H2O2 and L‑histidine combination. The combination of 100 mM H2O2 and 1.0% L‑histidine significantly reduced the number of viable cells of extended‑spectrum‑β‑lactamase‑producing E. coli and multidrug‑resistant Pseudomonas aeruginosa, and this treatment was more effective than 100 mM H2O2 alone, but this effect was not observed in methicillin‑resistant Staphylococcus aureus or vancomycin‑resistant Enterococcus faecium. The combination of L‑histidine and H2O2 may be a useful strategy to selectively increase the microbicidal activity of oxidative agents against Gram‑negative bacteria.

Introduction

Aerobically growing bacteria utilize oxygen species for energy metabolism. Reactive oxygen species (ROS), including superoxide anions (O2) and hydrogen peroxide (H2O2), are generated during this process (1,2). These ROS impair cellular functions by damaging DNA, proteins, and lipid membranes. Aerobic bacteria have evolved oxidative stress responses, including enzymes to scavenge ROS (catalases, superoxide dismutases and alkyl hydroperoxide reductases) (3,4) and the rec system for DNA repair (5). However, oxidative stress that overwhelms the capacity of these stress response systems kills the bacteria. Therefore, oxidative antimicrobials are widely used for sanitation purposes in healthcare facilities.

A compromised DNA repair system has been reported to render microbes vulnerable to oxidative stress (3,5). In the present study, L-histidine suppressed the proliferation of recA-deficient Escherichia coli laboratory strains under aerobic conditions, but not anaerobic conditions. This indicated that L-histidine enhances the oxidative DNA damage of E. coli cells. L-histidine has been reported to enhance the genotoxicity of hydrogen peroxide (H2O2) in mammalian cells by increasing ROS production (69).

H2O2 is produced in hostile eukaryotic hosts (10) or from resident microbes (11), and eradicates pathogenic bacteria or competitors in the same habitat, respectively. H2O2 is a non-polar molecule that penetrates lipid membranes and oxidizes intracellular molecules (12,13). H2O2 generates hydroxyl radicals (OH·) through reacting with iron in a process known as the Fenton reaction (14). OH· is a powerful oxidant that reacts with a wide range of organic substances (1).

In previous decades, the microbicidal activity of H2O2 has been utilized for wastewater disinfection and the sanitation of hospital environments. For example, H2O2 vapor is used for bedroom sanitation in hospitals, and treatment of rooms occupied by patients colonized with multidrug-resistant microorganisms (MDROs) reduces the risk of carriage of these MDROs by 64% (15). In addition, a combination of H2O2 and low concentrations of anionic and/or nonionic surfactants has been commercialized as a disinfectant for healthcare settings (16) H2O2 is recognized as an environment-friendly reagent that does not leave unfavorable environmental effects (1719).

The main disinfection mechanism of H2O2 involves hydroxyl or hydroperoxyl radical production (20). The combination of H2O2 and metallic ions, including ferrous iron (Fe2+) or copper (Cu2+) enhances the disinfection potential via the Fenton reaction, or Fenton-like reactions (21). H2O2 combined with copper or silver synergistically enhances the disinfection efficiency of wastewater (22,23). In addition, modifications of the disinfection efficacy of H2O2 through combination with other components have been reported, including surfactant (16), chlorhexidine (24), iodine (25), sodium bicarbonate (26), hypothiocyanate (27), rifampicin (28), organic acids (29), neucopropine (30), and UV-irradiation (31). In the present study, L-histidine was also demonstrated to augment the bactericidal activity of H2O2 against Gram-negative bacteria.

Materials and methods

Reagents

H2O2 (50% w/v in H2O) and L-histidine were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and Wako Chemicals GmbH (Neuss, Germany), respectively.

Bacterial strains and culture conditions

Laboratory strains of E. coli MG1655 (American Type Culture Collection, Manassas, VA, USA), JM109 (New England BioLabs, Inc., Ipswich, MA, USA), and DH5α (Thermo Fisher Scientific, Inc., Waltham, MA, USA), Pseudomonas aeruginosa PAO1 (Japan Collection of Microorganisms, Tsukuba, Ibaraki, Japan) and clinical isolates of multidrug resistant P. aeruginosa (MDRP) TUP1 from human blood (Tokushima University Hospital, Tokushima, Japan), methicillin-resistant Staphylococcus aureus (MRSA) TUM1 from human sputum (Tokushima University Hospital), extended-spectrum β-lactamase (ESBL)-producing E. coli KUM1 (corresponding to the isolate E6 reported by Uemura et al (Kagawa University Hospital, Miki, Japan) (32), and vancomycin-resistant Enterococcus faecium (VRE) FN1 (Dr Koichi Tanimoto, Gunma University, Maebashi, Japan) (33) were used in the present study. Glycerol stocks (−70°C) of these strains were streaked onto brain heart infusion (BHI; Eiken Chemical Co., Ltd., Tokyo, Japan) agar plates, which were incubated at 37°C for 24 h in the dark. A single colony from the BHI agar plate was inoculated into 3 ml BHI broth and incubated aerobically at 37°C for 16 h. Then, 1 ml of the culture was centrifuged (10,000 × g, 5 min, 4°C), and the collected cells were washed once with 2.5 ml phosphate-buffered saline (PBS; pH 7.4). The bacterial cells were then suspended in 4 ml PBS and used as the inoculum in the bactericidal test. Ten-fold serial dilutions of bacterial suspension were prepared and 0.1 ml of this suspension was spread on BHI agar plates to enumerate the viable cell counts in the suspension.

Monitoring of E. coli cell proliferation in the presence of L-histidine

The E. coli K12 strain MG1655 and the recA-deficient strains JM109 and DH5α were cultured in 3 ml BHI with shaking at 37°C overnight in the dark. Each overnight culture (30 μl) was used to inoculate two sets of 3 ml BHI supplemented with or without 1% L-histidine. One of the tubes was grown aerobically with shaking at 37°C in the dark, and the other was grown anaerobically at 37°C in the dark, using an anaerobic chamber conditioned with mixed gas (N2, 80%; H2, 10%; CO2, 10%). The proliferation of E. coli strains was monitored every 2 h by checking the optical density at 600 nm (OD600) until 10 h following inoculation.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of recA genes

Total RNA was extracted using the hot-phenol method (34) from the mid-logarithmic-phase cultures (OD660, 0.4–0.6) of E. coli MG1655 strains with or without exposure to 10 mM H2O2 (30 min at 37°C) and/or 1% L-histidine in the dark. The RNA was further purified using the RNeasy CleanUp kit (Qiagen GmbH, Hilden, Germany) and was treated with TURBO DNA-free (Ambion; Thermo Fisher Scientific, Inc.) to remove contaminating DNA. Total RNA (400 ng) was reverse transcribed using a PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan) with random hexamers at 37°C for 15 min. Reverse transcription was terminated by heating the mixture at 85°C for 5 sec. The cDNA products were subsequently amplified using SYBR Premix Ex Taq II (Takara Bio, Inc.) under the following conditions: Preheating at 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec in a StepOne Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The oligonucleotide primers used for monitoring recA expression were as follows: Forward, 5′-GTGAAGAACAAAATCGCTGC-3′ and reverse, 5′-TCTGCTACGCCTTCGCTAT-3′ (35). All samples were run in triplicate. Threshold cycle values were normalized to the levels of rrnH transcripts, and changes were calculated using the 2−ΔΔCq method (36). The PCR primers used for rrnH were as follows: Forward, 5′-AGTCGAACGGTAACAGGAAGA-3′ and reverse, 5′-GCAATATTCCCCACTGCTG-3′.

Assessment of DNA damage

E. coli MG1655 were statically incubated in 10 ml BHI for 16 h at 37°C in the dark. The culture was centrifuged (10,000 × g, 5 min, 4°C) and suspended in 10 ml PBS (pH 7.4) or PBS containing 1% L-histidine. H2O2 was added to the tubes (1, 10 or 100 mM) and incubated for 30 min at 37°C in the dark. Distilled water was used as a control in place of H2O2. Following incubation, chromosomal DNA fragmentation of the treated E. coli cells was assessed by pulsed-field gel electrophoresis (PFGE) as follows: Treated cells were embedded into 1% agarose to make sample plugs. The E. coli cells were then lysed within the agarose plug using the CHEF Bacterial DNA Genome kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. Genomic DNA was prepared in situ in agarose blocks. The DNA was resolved by PFGE in 1% agarose (SeaKem GTG agarose, Lonza Japan Ltd., Tokyo, Japan) in 0.5x Tris-borate-EDTA buffer with a CHEF-DR II system (Bio-Rad Laboratories, Inc.) at 14°C under 6 V/cm of electric field. Pulse time was set to 1–30 sec for the first 17 h, and changed to 5–9 sec for the last 6 h. Following electrophoresis, the gel was stained with 0.5 μg/ml ethidium bromide for 20 min at room temperature and washed with distilled water. The length of the smear was compared between samples treated with distilled water or H2O2 (1, 10 or 100 mM) with or without 1% L-histidine.

Measurement of ROS

Intracellular ROS levels were measured using the OxiSelect Intracellular ROS assay kit (Cell Biolabs, Inc., San Diego, CA, USA). E. coli MG1655 was cultured aerobically in 3 ml BHI to the mid-logarithmic phase and collected by centrifugation (10,000 × g, 5 min, 4°C). The cells were washed once with PBS and resuspended in PBS containing ROS substrate at 37°C for 60 min. The cells absorbing ROS substrate were collected, washed once with PBS and resuspended in PBS containing 2% L-histidine. An equal volume of 20 mM H2O2 was added to the cell suspension and incubated for 10 min at 30°C. Following incubation, 2′,7′-dichlorodihydrofluorescein (DCF) generated by the oxidative degradation of the substrate was monitored at 480 nm excitation/530 nm emission.

Free radical analysis using electron paramagnetic resonance (EPR) spectroscopy

Generation of free radicals in E. coli MG1655 following exposure to H2O2 and L-histidine was confirmed using an EPR-spin trapping method at room temperature. The spin trapping reagent, 5,5-dimethyl-1-pyrrorine-N-oxide (DMPO), was obtained from Labotec Co., Ltd. (Tokyo, Japan). The E. coli MG1655 cell suspension [0.1 ml; 5.6×108 colony forming units (CFU)] containing 2% L-histidine and 500 mM DMPO was incubated for 5 min at room temperature, and 0.1 ml 200 mM H2O2 was then added. After 5 min, the mixture was centrifuged at 2,000 × g for 1 min at room temperature and the cell pellets were resuspended in 0.1 ml PBS. EPR measurement was performed at room temperature. The sample was transferred to three sections of glass capillaries (10 μl; Drummond Scientific Co., Inc., Broomall, PA, USA) and set into the EPR cavity for the measurements. A Bruker EMXPlus EPR spectrometer (Bruker Corporation, Billerica, MA, USA) with an X-band cavity (ER 4103TM) was used to collect the EPR signal of the DMPO spin adducts. The typical instrumental conditions were as follows: 10 mW microwave power, 2.0-Gauss modulation amplitude, 0.08-sec time constant, 120-sec scan time, and 100-Gauss scan range. Hyperfine coupling constants and radical concentrations were obtained using the computer program Winsim (37).

H2O2 bactericidal test

The final concentrations of H2O2 used for the bactericidal test against Gram-negative (MDRP TUP1 and ESBL-producing E. coli KUM1) and Gram-positive (MRSA TUM1 and VRE FN1) bacteria were 100 and 200 mM, respectively. Each bacterial suspension (0.25 ml) was added to 0.5 ml of 2% histidine or sterilized distilled water. The suspension was mixed with 0.25 ml H2O2 (400 or 800 mM) and incubated at 25°C for 15 min. Following incubation, the mixture (15 μl) was transferred to 1.5 ml catalase solution (10 mg/ml; Sigma-Aldrich; Merck KGaA) to inactivate H2O2. Serial 10-fold dilutions of the mixture were prepared and 0.1 ml of each dilution was spread onto BHI plates in triplicate. Following incubation of the plates at 37°C for 24 h, the number of surviving cells was calculated from the colony counts grown on the BHI plates.

Statistical analysis

Data are expressed as the mean ± standard deviation. Statistical analysis of the data was performed with StatFlex 6.0 (Artech Co., Ltd., Osaka, Japan) using one-way analysis of variance to compare the means of all groups, followed by Tukey's test to compare the means of two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of L-histidine on the aerobic proliferation of recA-deficient E. coli strains

The MG1655 E. coli strain and the recA-deficient strains JM109 and DH5α were cultured in BHI broth supplemented with 1% L-histidine. As presented in Fig. 1, E. coli MG1655 grew well in the presence of 1% L-histidine under aerobic and anaerobic conditions. In contrast, JM109 and DH5α did not grow under aerobic conditions in the presence of 1% L-histidine, while they were able to grow under anaerobic conditions in the presence of this amino acid. As RecA plays a crucial role in DNA repair, these results indicated that L-histidine may induce oxidative DNA damage in the presence of oxygen, resulting in a lethal effect on the bacterial strains with impairments in the DNA repair system.

SOS response in E. coli induced by H2O2

DNA damage induces the expression of SOS stress response genes in order to repair the injury. The recA gene is a representative SOS stress response gene. To assess the level of DNA damage induced by H2O2 in the presence of L-histidine, recA gene expression in aerobically grown, mid-logarithmic phase E. coli MG1655 cells was measured by RT-qPCR (Fig. 2). The relative recA expression levels following treatment with 10 mM H2O2 increased ~5-fold compared with the distilled water control. The combination treatment with 10 mM H2O2 and 1% L-histidine induced a ~7-fold increase in recA gene expression compared with 10 mM H2O2 alone. Treatment with 1% L-histidine alone did not increase recA expression.

Assessment of DNA damage following exposure to H2O2

DNA damage induced by H2O2 and by the H2O2/L-histidine combination in statically grown E. coli MG1655 cells was assessed by PFGE. As presented in Fig. 3, treatment of E. coli MG1655 cells with 1% L-histidine alone did not induce DNA damage. DNA degradation was not observed in the E. coli cells treated with 1 mM H2O2 regardless of the presence of L-histidine. In contrast, a clear difference in DNA degradation appeared when >10 mM H2O2 was used, as E. coli DNA was degraded more extensively when 1% L-histidine was combined with 10 or 100 mM H2O2 compared with H2O2 treatment alone.

ROS generation in E. coli cells following exposure to H2O2

Intracellular ROS levels in E. coli MG1655 cells grown to mid-logarithmic phase under aerobic conditions and exposed to H2O2 were compared in conditions with or without 1% L-histidine. As presented in Fig. 4, intracellular ROS levels were low without H2O2. Addition of 1% L-histidine alone did not induce ROS generation. In contrast, exposure to 10 mM H2O2 significantly induced ROS generation in E. coli MG1655 cells compared with the negative control. In addition, ROS levels in E. coli significantly increased when the cells were treated with a combination of 10 mM H2O2 and 1% L-histidine compared with the other groups.

EPR analysis

EPR analysis was performed to identify which types of free radical were generated in E. coli cells following exposure to 100 mM H2O2 and 1% L-histidine. As presented in Fig. 5A, a typical four-line EPR signal attributed to DMPO-OH spin-adduct with an intensity ratio of 1:2:2:1 (hyperfine splitting constant; aN=15.300 and aH=14.549 gauss) (38) was observed by Fenton reaction systems containing 20 mM Fe2+, 100 mM H2O2 and 500 mM DMPO. The EPR spectrum representing DMPO-OH was observed in E. coli cells exposed to 100 mM H2O2 and 1% L-histidine (Fig. 5B). On the other hand, this EPR signal was not observed in E. coli cells exposed to 100 mM H2O2 alone (Fig. 5C). These data indicated that L-histidine promoted intracellular hydroxyl radical formation by H2O2 in E. coli cells.

Effect of L-histidine on the bactericidal activity of H2O2 against Gram-positive MDROs

To examine the sensitivity of MRSA and VRE to H2O2, aerobically-grown overnight cultures of these MDROs were exposed to 100, 200 and 300 mM H2O2 for 15 min. As presented in Fig. 6, H2O2 reduced the number of viable MRSA cells in a dose-dependent manner, and 100, 200 and 300 mM H2O2 achieved reductions of 0.29±0.07, 0.58±0.27 and 1.15±0.85 log10 CFU/ml in MRSA. H2O2 also reduced the viable numbers of VRE cells dose-dependently, and 100, 200 and 300 mM H2O2 achieved reductions of 0.31±0.19, 1.94±1.22 and 2.97±1.40 log10 CFU/ml in VRE.

Next, the effect of the combination of L-histidine and 200 mM H2O2 on the viability of these Gram-positive bacteria was assessed. As presented in Table I, no bactericidal effect was observed in the presence of 1% L-histidine alone. Addition of 200 mM H2O2 alone resulted in 0.89±0.35 and 1.22±0.74 log10 CFU/ml reductions in MRSA and VRE, while 200 mM H2O2 with 1% L-histidine reduced cell numbers only by 0.40±0.14 and 0.92±0.63 log10 CFU/ml, respectively. Thus, L-histidine did not improve the bactericidal effect of H2O2 against MRSA and VRE.

Table I

Effect of L-histidine on the bactericidal activity of H2O2 against multidrug-resistant microorganisms.

Table I

Effect of L-histidine on the bactericidal activity of H2O2 against multidrug-resistant microorganisms.

Treatment
Organisms (Log10 CFU/ml reduction)
H2O2 (mM)1% L-histidineMRSAVREMDRPESBL E. coli
+−0.01±0.13a,b−0.01±0.09a,b0.00±0.12d,e−0.07±0.09d,e
100NTNT0.24±0.16e,f0.76±0.08e,f
+NTNT1.12±0.36d,f1.14±0.02d,f
2000.89±0.35b,c1.21±0.74cNTNT
+0.40±0.14a,c0.91±0.63cNTNT

{ label (or @symbol) needed for fn[@id='tfn1-ijmm-41-05-2847'] } Data are presented as the mean ± standard deviation, obtained from three independent experiments. NT, not tested; MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-resistant Enterococcus faecium; MDRP, multidrug resistant P. aeruginosa; ESBL, extended-spectrum β-lactamase.

a P<0.01 vs. 200 mM H2O2,

b P<0.01 vs. 200 mM H2O2+1% L-histidine,

c P<0.01 vs. 1% L-histidine,

d P<0.01 vs. 100 mM H2O2,

e P<0.01 vs. 100 mM H2O2+1% L-histidine,

f P<0.01 vs. 1% L-histidine.

Effect of L-histidine on the bactericidal activity of H2O2 against Gram-negative MDROs

The bactericidal activities of 100 mM H2O2 against aerobically grown overnight cultures of MDRP or ESBL-producing E. coli were assessed in the presence or absence of 1% histidine (Table I). H2O2 alone achieved 0.24±0.16 and 0.76±0.08 log10 CFU/ml reductions in MDRP and ESBL-producing E. coli cells, respectively. Addition of 100 mM H2O2 along with 1% L-histidine reduced cell counts by 1.12±0.36 and 1.14±0.02 log10 CFU/ml in these bacteria, respectively. These results indicated that L-histidine significantly enhanced the bactericidal action of H2O2 against MDRP and ESBL-producing E. coli (P<0.01).

Discussion

In the present study, L-histidine supplementation in the culture media was revealed to suppress the proliferation of the recA-deficient E. coli laboratory strains JM109 and DH5α, but this amino acid did not influence the proliferation of strain MG1655, which possesses an intact rec system. In addition, this suppression was not apparent when the strains were cultured anaerobically. These results indicate that L-histidine may increase oxidative DNA damage to E. coli, because RecA serves a crucial function in DNA repair. This hypothesis was supported by the experimental data, which revealed that recA gene expression in E. coli MG1655 cells exposed to 10 mM H2O2 plus 1% L-histidine increased 7-fold compared with that in cells exposed to 10 mM H2O2 alone. In addition, DNA degradation was more evident in the E. coli cells exposed to 10–100 mM H2O2 plus 1% L-histidine than in cells treated with the same concentration of H2O2 alone.

The augmenting effect of L-histidine on H2O2-induced DNA damage may be mediated by a Fenton-like reaction initiated by the contact of oxidative agents with metal ions, generating ROS (21). Since the imidazole group in L-histidine is known to bind to metal ions, including Ni+ or Co+, this characteristic has been applied to histidine-tagged recombinant protein purification. Prior work has demonstrated that L-histidine increased the genotoxicity of H2O2 against Chinese hamster ovary (CHO) cells (8,9) and that this is dependent on the L-histidine transporting activity of the CHO cells (39). Therefore, metal ions may be co-transported inside the E. coli cells with L-histidine, and these metal ions may increase the H2O2-derived DNA damage by a Fenton-like reaction. In fact, our group detected hydroxyl radicals in E. coli cells following treatment with H2O2 and L-histidine, but an EPR signal was not observed when the cells were treated with H2O2 alone. Schubert et al (7) reported that L-hisitidine and H2O2 readily form a stable adduct. Their group indicated that L-hisitidine-peroxide adduct may enter bacterial cells more rapidly than H2O2 alone, or that the rate of breakdown of the adduct by catalase is slower.

Consistent with the augmenting effect of L-histidine on H2O2-derived DNA damage in E. coli, L-histidine increased the killing activity against Gram-negative MDROs: ESBL-producing E. coli and MDRP. Wide penetration of multidrug resistance (MDR) in Gram-negative bacilli is a serious, global public health concern (40). MDR in highly virulent bacteria including fluoroquinolone-resistant Salmonella (41) or ESBL-producing Shiga-toxigenic E. coli (42,43) is emerging. In addition, the dissemination of carbapenemase-producing Enterobacteriaceae has made it difficult to treat certain infectious diseases with antibiotics (44). Thus, transmission control of Gram-negative bacilli with MDR is an urgent issue. Disinfection techniques using a combination of H2O2 and L-histidine may contribute to the efficient eradication of these MDR Gram-negative bacteria from hospital environments. However, H2O2 is a highly reactive oxidizing agent and has genotoxic effects. Oosterik et al (45) reported that nebulization of 2% H2O2 renders chickens more susceptible to avian pathogenic E. coli, potentially due to epithelial damage by hydroxyl radicals. The simultaneous use of L-histidine may decrease the H2O2 concentration necessary to eradicate Gram-negative pathogens.

In contrast to Gram-negative MDROs, L-histidine did not increase the microbicidal activity of H2O2 against Gram-positive bacteria (MRSA and VRE). The reason for this selective augmentation remains unclear but L-histidine transporting activity may be different between Gram-negative and Gram-positive bacteria. This selective effect of L-histidine on the bactericidal activity of H2O2 may be advantageous in certain instances, including during the elimination of foodborne pathogens (for example, Salmonella and pathogenic E. coli from fermented food made with lactic acid bacteria) or for bioremediation of contaminated soil by Gram-positive bacteria. The physiological benefit of resident microflora for human health has also been increasingly recognized. For example, a resident skin microbe, Staphylococcus epidermidis, produces an Esp protease that degrades S. aureus biofilms, thereby conferring colonization resistance to MRSA (46). Thus, the use of the H2O2/L-histidine combination may be applicable for treatment of skin lesions, including decubitus ulcers infected with P. aeruginosa. H2O2/L-histidine is expected to exert enhanced toxicity against Gram-negative pathogens, while having a less detrimental effect on colonization resistance by normal resident skin flora.

In the present study, our group demonstrated the selective augmenting effect of L-histidine on the microbicidal activity of H2O2 against Gram-negative bacteria. This effect is potentially derived from DNA damage by ROS generated through Fenton-like reactions between H2O2 and metal ions bound to the imidazole group of L-histidine. The H2O2/L-histidine combination reduces the H2O2 concentration necessary to inactivate Gram-negative pathogens. In addition, this selectivity to Gram-negative bacteria may be useful in sanitation processes required to protect Gram-positive bacteria, including lactic acid bacteria in fermented foods or resident skin microbiota. Taken together, the results of the present study may provide valuable information to develop novel H2O2-based disinfection techniques.

Acknowledgments

The authors would like to thank Dr Koichi Tanimoto (Gunma University, Maebashi, Japan) for the gift of E. faecium FN1, and Mr. Hitoshi Yamaoka (Honbu Sankei Co. Ltd., Osaka, Japan) for technical assistance.

Notes

[1] Funding

The present study was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science KAKEN (grant no. 16K12012).

[2] Availability of data and materials

Data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study.

[3] Authors' contributions

TN and HNI analyzed and interpreted the effect of the test reagents on E. coli growth. AT and HNI performed recA qPCR and ROS measurement. ME and HY performed PFGE analysis. MH and KT performed EPR analysis. TN, ET and KO examined the bactericidal effect of test reagents. TN and TK were major contributors in writing the manuscript. All authors read and approved the final manuscript.

[4] Ethics approval and consent to participate

Not applicable.

[5] Consent for publication

Not applicable.

[6] Competing interests

The authors declare that they have no competing interests.

References

1 

Imlay JA: The molecular mechanisms and physiological consequences of oxidative stress: Lessons from a model bacterium. Nat Rev Microbiol. 11:443–454. 2013. View Article : Google Scholar : PubMed/NCBI

2 

Iuchi S and Weiner L: Cellular and molecular physiology of Escherichia coli in the adaptation to aerobic environments. J Biochem. 120:1055–1063. 1996. View Article : Google Scholar : PubMed/NCBI

3 

Gort AS and Imlay JA: Balance between endogenous superoxide stress and antioxidant defenses. J Bacteriol. 180:1402–1410. 1998.PubMed/NCBI

4 

Park S, You X and Imlay JA: Substantial DNA damage from submicromolar intracellular hydrogen peroxide detected in Hpx mutants of Escherichia coli. Proc Natl Acad Sci USA. 102:9317–9322. 2005. View Article : Google Scholar

5 

Touati D, Jacques M, Tardat B, Bouchard L and Despied S: Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: Protective role of superoxide dismutase. J Bacteriol. 177:2305–2314. 1995. View Article : Google Scholar : PubMed/NCBI

6 

Oya Y and Yamamoto K: The biological activity of hydrogen peroxide. IV. Enhancement of its clastogenic actions by co-administration of L-histidine. Mutat Res. 198:233–240. 1988. View Article : Google Scholar : PubMed/NCBI

7 

Schubert J, Watson JA and Baecker JM: Formation of a histidine-peroxide adduct by H2O2 or ionizing radiation on histidine: Chemical and microbiological properties. Int J Radiat Biol Relat Stud Phys Chem Med. 14:577–583. 1969. View Article : Google Scholar : PubMed/NCBI

8 

Sestili P, Cattabeni F and Cantoni O: The L-histidine-mediated enhancement of hydrogen peroxide-induced DNA double strand breakage and cytotoxicity does not involve metabolic processes. Biochem Pharm. 50:1823–1830. 1995. View Article : Google Scholar : PubMed/NCBI

9 

Tachon P, Deflandre A and Giacomoni PU: Modulation by L-histidine of H2O2-mediated damage of cellular and isolated DNA. Carcinogenesis. 15:1621–1626. 1994. View Article : Google Scholar : PubMed/NCBI

10 

McRipley RJ and Sbarra AJ: Role of the phagocyte in host-parasie interactions. XII. Hydrohgen peroxide-myeloperoxidase bactericidal system in the phagocyte. J Bacteriol. 94:1425–1430. 1967.PubMed/NCBI

11 

Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, Critchlow CM and Holmes KK: Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol. 27:251–256. 1989.PubMed/NCBI

12 

Seaver LC and Imlay JA: Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol. 183:7173–7181. 2001. View Article : Google Scholar : PubMed/NCBI

13 

Seaver LC and Imlay JA: Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli. J Bacteriol. 183:7182–7189. 2001. View Article : Google Scholar : PubMed/NCBI

14 

Walling C: Fenton's reagent revisited. Accounts Chem Res. 8:125–131. 1975. View Article : Google Scholar

15 

Passaretti CL, Otter JA, Reich NG, Myers J, Shepard J, Ross T, Carroll KC, Lipsett PL and Perl TM: An evaluation of environmental decontamination with hydrogen peroxide vapor for reducing the risk of patient acquisition of multidrug-resistant organisms. Clin Infect Dis. 56:27–35. 2013. View Article : Google Scholar

16 

Rutala WA, Gergen MF and Weber DJ: Efficacy of improved hydrogen peroxide against important healthcare-associated pathogens. Infect Control Hosp Epidemiol. 33:1159–1161. 2012. View Article : Google Scholar : PubMed/NCBI

17 

Ronen Z, Guerrero A and Gros A: Graywater disinfection with the environmentally friendly hydrogen peroxide plus (HPP). Chemosphere. 78:61–65. 2010. View Article : Google Scholar

18 

Tang WZ and Tassos S: Oxidation kinetics and mechanisms of trihalomethanes by Fenton's reagent. Water Research. 31:1117–1125. 1997. View Article : Google Scholar

19 

Yu Y, Chan WI, Liao PH and Lo KV: Disinfection and solubilization of sewage sludge using the microwave enhanced advanced oxidation process. J Hazard Mater. 181:1143–1147. 2010. View Article : Google Scholar : PubMed/NCBI

20 

Oya Y, Yamamoto K and Tonomura A: The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts. Mutat Res. 172:245–253. 1986. View Article : Google Scholar : PubMed/NCBI

21 

Jung YS, Lim WT, Park JY and Kim YH: Effect of pH on Fenton and Fenton-like oxidation. Environ Technol. 30:183–190. 2009. View Article : Google Scholar : PubMed/NCBI

22 

Aslani H, Nabizadeh R, Alimohammadi M, Mesdaghinia A, Nadafi K, Nemati R and Ghani M: Disinfection of raw wastewater and activated sludge effluent using Fenton like reagent. J Environ Health Sci Eng. 12:1492014. View Article : Google Scholar

23 

Orta De Velásquez MT, Yáñez-Noguez I, Jiménez-Cisneros B and Luna Pabello VM: Adding silver and copper to hydrogen peroxide and peracetic acid in the disinfection of an advanced primary treatment effluent. Environ Technol. 29:1209–1217. 2008. View Article : Google Scholar : PubMed/NCBI

24 

Steinberg D, Heling I, Daniel I and Ginsburg I: Antibacterial synergistic effect of chlorhexidine and hydrogen peroxide against Streptococcus sobrinus, Streptococcus faecalis and Staphylococcus aureus. J Oral Rehabil. 26:151–156. 1999. View Article : Google Scholar : PubMed/NCBI

25 

Zubko EI and Zubko MK: Co-operative inhibitory effects of hydrogen peroxide and iodine against bacteria and yeast species. BMC Res Notes. 6:2722013. View Article : Google Scholar

26 

Miyasaki KT, Genco RJ and Wilson ME: Antimicrobial properties of hydrogen peroxide and sodium bicarbonate individually and in combination against selected oral, Gram-negative, facultative bacteria. J Dent Res. 65:1142–1148. 1986. View Article : Google Scholar : PubMed/NCBI

27 

Carlsson J, Edlund MB and Hanstrom L: Bactericidal and cytotoxic effects of hypothiocyanite-hydrogen peroxide mixtures. Infect Immun. 44:581–586. 1984.PubMed/NCBI

28 

Humphrey TJ: The synergistic inhibition of Campylobacter jejuni by rifampicin and hydrogen peroxide. Lett Apple Microbiol. 10:97–100. 1990. View Article : Google Scholar

29 

Martin H and Maris P: Synergism between hydrogen peroxide and seventeen acids against six bacterial strains. J Appl Microbiol. 113:578–590. 2012. View Article : Google Scholar : PubMed/NCBI

30 

Almeida CE, Felício DL, Galhardo RS, Cabral-Neto JB and Leitão AC: Synergistic lethal effect between hydrogen peroxide and neocuproine (2,9-dimethyl 1,10-phenanthroline) in Escherichia coli. Mutat Res. 433:59–66. 1999. View Article : Google Scholar : PubMed/NCBI

31 

Shama G: Inactivation of Escherichia coli by ultraviolet light and hydrogen peroxide in a thin film contactor. Lett Appl Microbiol. 15:259–260. 1992. View Article : Google Scholar

32 

Uemura M, Imataki O, Uchida S, Nakayama-Imaohji H, Ohue Y, Matsuka H, Mori H, Dobashi H, Kuwahara T and Kadowaki N: Strain-specific transmission in an outbreak of ESBL-producing Enterobacteriaceae in the hematooncology care unit: A cohort study. BMC Infect Dis. 17:262017. View Article : Google Scholar

33 

Fujita N, Yoshimura M, Komori T, Tanimoto K and Ike Y: First report of the isolation of high-level vancomycin-resistant Enterococcus faecium from a patient in Japan. Antimicrob Agents Chemother. 42:25101998.

34 

Rocha ER and Smith CJ: Regulation of Bacteroides fragilis katB mRNA by oxidative stress and carbon limitation. J Bacteriol. 179:7033–7039. 1997. View Article : Google Scholar : PubMed/NCBI

35 

Kim YS, Min J, Hong HN, Park JH, Park KS and Gu MB: Gene expression analysis and classification of mode of toxicity of polycyclic aromatic hydrocarbons (PAHs) in Escherichia coli. Chemosphere. 66:1243–1248. 2007. View Article : Google Scholar

36 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Method. 25:402–408. 2001. View Article : Google Scholar

37 

Oya-Ohta Y, Ueda A, Ochi T, Harada M and Yamamoto K: The biological activity of hydrogen peroxide VII. L-Histidine increases incorporation of H(2)O(2) into cells and enhances formation of 8-oxodeoxyguanosine by UV-C plus H(2)O(2) but not by H(2)O(2) alone. Mutat Res. 478:119–127. 2001. View Article : Google Scholar : PubMed/NCBI

38 

Duling DR: Simulation of multiple isotropic spin-trap EPR spectra. J Magn Reson B. 104:105–110. 1994. View Article : Google Scholar : PubMed/NCBI

39 

Buettner GR: Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med. 3:259–303. 1987. View Article : Google Scholar : PubMed/NCBI

40 

Kaye KS and Pogue JM: Infections caused by resistant gram-negative bacteria: Epidemiology and management. Pharmacotherapy. 35:949–962. 2015. View Article : Google Scholar : PubMed/NCBI

41 

Sjölund-Karlsson M, Folster JP, Pecic G, Joyce K, Medalla F, Rickert R and Whichard JM: Emergence of plasmid-mediated quinolone resistance among non-typhi Salmonella enterica isolates from humans in the United States. Antimicrob Agents Chemother. 53:2142–2144. 2009. View Article : Google Scholar : PubMed/NCBI

42 

Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth A, Bauwens A, Peters G and Karch H: Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: A microbiological study. Lancet Infect Dis. 11:671–679. 2011. View Article : Google Scholar : PubMed/NCBI

43 

Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y, Zhang W, et al: Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One. 6:e227512011. View Article : Google Scholar : PubMed/NCBI

44 

Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT and Daikos GL: Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: An evolving crisis of global dimensions. Clin Microbiol Rev. 25:682–707. 2012. View Article : Google Scholar : PubMed/NCBI

45 

Oosterik L, Tuntufye HN, Janssens S, Butaye P and Goddeeris BM: Disinfection by hydrogen peroxide nebulization increases susceptibility to avian pathogenic Escherichia coli. BMC Res Notes. 8:3782015. View Article : Google Scholar : PubMed/NCBI

46 

Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, Agata T and Mizunoe Y: Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature. 465:346–349. 2010. View Article : Google Scholar : PubMed/NCBI

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May-2018
Volume 41 Issue 5

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Online ISSN:1791-244X

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Spandidos Publications style
Nagao T, Nakayama‑Imaohji H, Elahi M, Tada A, Toyonaga E, Yamasaki H, Okazaki K, Miyoshi H, Tsuchiya K, Kuwahara T, Kuwahara T, et al: L‑histidine augments the oxidative damage against Gram‑negative bacteria by hydrogen peroxide. Int J Mol Med 41: 2847-2854, 2018
APA
Nagao, T., Nakayama‑Imaohji, H., Elahi, M., Tada, A., Toyonaga, E., Yamasaki, H. ... Kuwahara, T. (2018). L‑histidine augments the oxidative damage against Gram‑negative bacteria by hydrogen peroxide. International Journal of Molecular Medicine, 41, 2847-2854. https://doi.org/10.3892/ijmm.2018.3473
MLA
Nagao, T., Nakayama‑Imaohji, H., Elahi, M., Tada, A., Toyonaga, E., Yamasaki, H., Okazaki, K., Miyoshi, H., Tsuchiya, K., Kuwahara, T."L‑histidine augments the oxidative damage against Gram‑negative bacteria by hydrogen peroxide". International Journal of Molecular Medicine 41.5 (2018): 2847-2854.
Chicago
Nagao, T., Nakayama‑Imaohji, H., Elahi, M., Tada, A., Toyonaga, E., Yamasaki, H., Okazaki, K., Miyoshi, H., Tsuchiya, K., Kuwahara, T."L‑histidine augments the oxidative damage against Gram‑negative bacteria by hydrogen peroxide". International Journal of Molecular Medicine 41, no. 5 (2018): 2847-2854. https://doi.org/10.3892/ijmm.2018.3473