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Virology & Immunology Journal Research Article 13 min read

Mechanism of Antibacterial Activities of Cu (â…¡) Ions against Staphylococcus aureus and Escherichia coli on the Ground of Results Obtained from Dilution Medium Method

Ishida T*
* Corresponding author
ISSN: 2577-4379  10.23880/vij-16000117  Received: September 22, 2017  Published: October 05, 2017
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Keywords
MIC MBC CFU measurements Cu2+ ions PGN cell wall Outer membrane lipoproteins Biosynthesis and autolysin Reactive oxygen species (ROS)
Abstract

Antibacterial activities by copper(Ⅱ) ion solution based antibacterial results of broth dilution medium method were investigated. From dilution medium method, Minimum Inhibitory Concentration (MIC) = 625 mg/L as bacteriostatic action, Minimum Bactericide Concentration (MBC) = 1250 mg/L as bactericide action by Cu2+ ion solution were obtained against Staphylococcus aureus. Bacteriolysis of Staphylococcus aureus peptidoglycan (PGN) cell wall by Cu2+ ions is ascribed to the inhibition of PGN elongation due to the damages of PGN biosynthesis of transglycosylase (TG) and transpeptidase (TP), and the activations of PGN forth autolysins. The other, bacteriolysis of Escherichia coli outer membrane cell wall by Cu2+ ions is attributed to the destruction of outer membrane structure and to the inhibition of PGN elongation due to the damage of PGN biosynthesis TP and the activations of PGN autolysins. Cu2+ ions induced ROS such as O2－, H2O2,･OH, OH－ producing in bacterial cell wall occur oxidative stress.

Introduction

Silver, copper, and zinc of transition metals have highly antibacterial activities and are utilized as chemotherapy agents. Recently, antibacterial activities of copper, zinc and these complexes call attention to potential treatments such as control of infectious diseases [1, 2], exploitation during bacterial pathogenesis [3, 4, 5], adaptive immune response to virus [6] and binds with viral proteins [7], Mechanism of Antibacterial Activities of Cu (Ⅱ) Ions against Staphylococcus aureus and Escherichia coli on the Ground of Results Obtained from Dilution Medium Method novel copper-based formulations on such as E.coli O157, Salmonella, Helicobacter pylori [8, 9], and regulation of cancer and tumor cells [10, 11]. Copper is required for the function of several important enzymes including the cytochrome oxidase, copper-zinc superoxidase. Copper is viral inhibitors with the commonest metal ion that binds to viral proteins. However, copper can also be extremely Virol Immunol J

toxic, requiring homeostatic mechanism. Research of Cu ions killing mechanism has been carried out that Cu ions induced lead to the instability of membrane fatty acid integrity and homeostasis, increased levels of lipid peroxidation against both Gram-negative and Gram- positive bacteria [12]. In this way, the detailed mechanisms by which copper ions enter the bacterial cells are scarcely known. In this study, the broth dilution medium method test against S.aureus and E.coli, were carried out, where in it was turned out that antibacterial effects of Cu2+ ion solution were examined. On the basis of the high antibacterial activities for these Cu2+ ions, the processes of bacteriolysis and destructions of bacterial cell walls by copper (Ⅱ) ions had been considered against S. aureus peptidoglycan (PGN) and E.coli outer membrane cell walls. Furthermore, the bacteriolytic mechanisms by copper (Ⅱ) ion have been also revealed against both S.aureus and E.coli bacteria.

Method

Two-fold Broth Dilution Medium Method Tests for Cu2+ Ion Solutions

This method is quantitatively obtained for the antibacterial activity on the bactericidal assay. Bacteria intended for two-fold broth dilution medium method were treated as Staphylococcus aureus (NBRC12732) and Escherichia coli (ATCC25922). The other, the antibacterial copper ion of commercial copper (Ⅱ) ion agent (Japan ion production Ltd., original Cu2+ ion solution; 500 mg/L) are used as bacteriostasis, and copper nitrate (Cu(NO3)23H2O,Wako Pure Reagents) of special class reagent was used as bactericide action. Firstly, the sample test tube of Cu2+ ion concentration of 10,000 mg/L have been prepared in heart infusion agar medium(Nissui). Next, the diluted solutions of 10-stagesby two-fold dilution solution method was adjusted in tenth sample tubes for Cu2+ ion solution concentration of 9.8～5,000 mg/L. Afterwards, the adjustment solution within final solution of 5×10５cfu/mL was prepared, and then with a sterile micropipette, fungous liquid 1 mL of bacterial suspension was respectively transferred from tube No 1 to other tubes that were inoculated into the respective tubes. Finally, the tubes were incubated at 35℃ for 24 hours, in which the incubated solutions were afforded to minimum inhibitory concentration (MIC), minimum bactericide concentration (MBC), colony forming unit (CFU) measurements.

Search and Analysis for Bacterial Cell Molecular Structure, and PGN Biosyntheses and Autolysins

The surface envelop cell structures of S.aureus as representative of Gram-positive bacterium and E.coli as representative of Gram-negative bacterium, molecular structures of these cell walls, molecular structure of peptidoglycan (PGN), and PGN biosyntheses and autolysins were searched in detail. Further, the reaction and the behavior of metallic ions and bacterial cell, molecular bonding manner, and copper ion characteristics were also searched.

Results

Bacteriostatic and Bactericide Actions for Cu2+ Ionsolution by the Broth Dilution Medium Method

Table 1 shows the bacteriostasis as disinfection agent inhibiting the bacteria growth and multiplying organism of Cu2+ ion, in which minimum inhibitory concentration, MIC=50 mg/L above was obtained for Cu2+ ion concentration range of 0.10～50 mg/L [13]. The other, Table 2 indicates the results as bactericide action, in which MIC=625 mg/L and minimum bactericide concentration, MBC=1250mg/L were obtained for Cu2+ ion concentration range of 9.8～5000 mg/L [14]. The killing curve of Cu2+ ions is shown in Fig.1 (measurement’s error=±6%), in which killing effects for the copper (Ⅱ) ions appear sufficiently.

Figure 1: Relationship between increasing Cu2+ concentration (mg/L) and viable counts (CFU/mL) against S.aureus.
Click to enlarge
Figure 1: Relationship between increasing Cu2+ concentration (mg/L) and viable counts (CFU/mL) against S.aureus.
MIC 50
mg/L
above
Cu2+ solution concentration(mg/L)
Cu2+ solution agent･
502512.56.253.131.560.780.390.20.1
original conc 500 mg/L
＋＋＋＋＋＋＋＋＋＋

Table 1: MIC measurements of commercial Cu2+ solution agents as a bacteriostatic action against E.coli by broth dilution medium m

(＋): Visible bacterial growth (－): No visible bacterial growth Table 1: MIC measurements of commercial Cu2+ solution agents as a bacteriostatic action against E.coli by broth dilution medium method.

Antibacterial agentCu2+ concentration(mg/L)
Cu(NO ) 3H O
3 2 2		5000250012506253131567839209.8
solution
MIC____++++++
－－－+++++++
MBC
＜10＜10＜101.1 ×
102		3.1 ×
108		4.0 ×
108		4.5 × 1085.1 ×
108		5.5 ×
108		5.3 ×
108
CFU(cfu/mL)

Table 2: MIC, MBC, and CFU of Cu2+ in Cu (NO3)23H2O solution as a bactericidal action against S.aureus by 10-fold diluted solutio

（＋）: Bacterial growth (visible turbidity), （－）: No visible bacterial growth Table 2: MIC, MBC, and CFU of Cu2+ in Cu (NO3)23H2O solution as a bactericidal action against S.aureus by 10-fold diluted solution medium method.

Results of Search and Analysis

S.aureus and E.coli Cell walls, Action Sites of PGN biosyntheses of transglycosylase TG and transpeptidase TP and PGN autolysins: S.aureus surface cell envelop consists of teichoic acids, lipoteichoic acids, and thick peptidoglycan (below PGN) cell wall [13], whereas E.coli cell wall comprised of lipid A, lipopoly- saccharide, porin proteins, outer membrane of lipoprotein, and thinner 2-7 nm PGN layer in 30-70 nm periplasmic space [15]. Figure 2 shows the molecular structure of S.aureus PGN cell wall, including the action sites of PGN biosynthesis enzymes of TG/TP, and PGN forth autolysins and Lysostaphin enzyme. Furthermore, Figure 3 represents the molecular structure of E.coli cell wall and periplasmic peptidoglycan, containing the action sites of the hydrolases of lipoproteins, the peptidoglycan biosynthetic enzymes TG/TP, and the autolysins. Further, interactions of PGN molecular structure, PGN syntheses andautolysins influence essentially in any event the bacteriolysis of bacterial cell walls.

Figure 2: The molecular structure of S.aureus PGN cell wall and the action sites of PGN biosynthesis enzymes of TG/TP, PGN forth autolysins, and Lysostaphin enzyme.
Click to enlarge
Figure 2: The molecular structure of S.aureus PGN cell wall and the action sites of PGN biosynthesis enzymes of TG/TP, PGN forth autolysins, and Lysostaphin enzyme.
Figure 3: Molecular structure of E.coli cell wall and periplasmic PGN, and the action sites of the hydrolases of LPT, the PGN synthetic enzymes TG/TP, and the autolysins.
Click to enlarge
Figure 3: Molecular structure of E.coli cell wall and periplasmic PGN, and the action sites of the hydrolases of LPT, the PGN synthetic enzymes TG/TP, and the autolysins.

Characteristics of Copper (Ⅱ) Ion Solution: In this experiment, Cu (Ⅱ) ion solution was used as antibacterial agent, in which the rapid killing of various bacteria in contact with metallic copper (Ⅱ) ion is thought to be influenced by the Cu (Ⅱ)/Cu (Ⅰ) redox reaction strength within the bacterial cell. Then, for example, by the reaction of Cu2+ ions with S.aureus surface, Cu2+-proteins may be formed, on the ground that is due to formation of S-atom containing Cu-cysteine complex in bacteria [16].

Discussions

Bacteriolysis of S.aureus PGN Cell Wall by Cu2+ Ions

Bacteriolysis by balance deletion between biosynthesis enzyme and decomposition enzyme (autolysin) in PGN cell wall: For the sake of growth of S.aureus PGN cell wall, there is necessarily required for the adequate balance between PGN biosynthesis and PGN autolysin. When the balance is broken by Cu2+ penetration, Cu2+ ions are self-catalytically treated as coenzyme, that this is indicated that activation of autolysin is preceded, in which bacteriolysis and killing may result. Hence, bacteriolysis of S.aureus PGN cell wall by Cu2+ions is thought to be due to inhibition of PGN elongation owing to the damages of PGN synthetic TG/TP [17] and the activations of PGN autolysin, AmiA [18, 19]. Inhibition of Polymerization of Glycan Chains Bonding and Cross-Linking of Side Peptide: Cu2+ ions inhibit polymerization of glycan chains, forming copper complex in which is partial action sites of glycan saccharide chains. L is coordinated molecular.

Cu2+ + LH → CuL+ + H+

CuL+ + LH → CuL2 + H+

Copper-complexes on saccharide chains may be ･―NAG-(NAM-Cu-2O-2N-NAG)-NAM―･ [20]. The other, Cu2+ ions inhibit cross-linked reaction by peptide copper complex formation bonding to side- peptide chains [21].

Cu2+ ＋ 2LH → CuL2 ＋ H+

Peptide copper complex may be 3N-Cu-O, Cu (Gly-L-Ala) H2O [20]. Specially, Cu2+ ions react with cross-molecular penta glycine (Gly)5, copper-glycine complex may be formed [22]. Amino acid：Cu2+ + Gly－→ Cu (Gly)＋, Cu (Gly)+ + Gly― → Cu(Gly)2, Peptido： Cu2+ + GlyGly→ Cu (GlyGly), Cu (GlyGly) + Gly―→Cu(GlyGlyGly)―.

Bacteriolysis and Destruction of E.coli Outer Membrane Cell Wall by Cu2+ Ions

Inhibition of outer membrane cell wall: Cu2+ ions inactivate catalyst enzyme with forming Cu+ ions.

Cu2+ + －SH → －SCu (Ⅰ) + H+ By the penetration of Cu2+ ions, as shown in Fig.3, the activations of amidase enzyme of N-terminal and endopeptidase enzyme of C-terminal are enhanced [23, 24]. Accordingly, the activations of decomposition at N-, C-terminals of lipoproteins may occur with the destruction of outer membrane structure.

Inhibition of Biosynthesis and Activation of Autolysin, or Regulation and Deletion of Autolysin: Inhibition of E.coli PGN by Cu2+ ions is reported [25], however, the site of concrete action is not described. In E.coli, it is unlikely thought that Cu2+ ions inhibit both TG and TP [26]. The other, it is unclear that Cu2+ inhibits the polymerization of NAM and NAG chains. It is perhaps simpler to think that TP enzyme of cross-linked reaction is inhibited by Cu2+ ions and the activation of PGN autolysin occurs. By the accumulation of Cu2+ ions in periplasmic space, it might be possible that bacteriolysis of cell wall occur by the activation of PGN autolysin within periplasmic space. Many autolysins of E.coli are regulated by metals ion such as Hg2+, Cu2+ [27]. This regulation or deletion of decomposition enzyme inhibits PGN elongation, in which the bacteriolysis of the cell wall is induced. These facts are consistent with that the destruction by bacteriolysis of cell wall had been observed against E.coli. Hence, bacteriolysis of E.coli cell wall by Cu2+ ions occurs by destruction of outer membrane structure due to degradation of lipoprotein at N-, C-terminals, damage of TP enzyme and activations of PGN autolysins [19]. Furthermore, deletion of PGN autolysin also becomes bacteriolystic factor. Antibacterial activities of cell membrane and cytoplasm: Copper in solution is present as cupric ions, Cu2+. The Cu2+ in bacteria is reduced to Cu1+ and that the cuprous ions are considerably more toxic and very unstable. In E.coli cell wall, reactive oxygen species (ROS) production occur by Cu2+ ion penetration.

－・ Cu2+ + O2

O2 + e → O2

－→ O2 + Cu+ O2

－ + e + 2H+ → H2O2 Cu2+ + H2O2 → Cu+ + H+ + HO2･ ROS O2

－and H2O2 generated in the cell wall, permeate into cell membrane and cytoplasm, in which high reactive･ OH and OH－ in cell membrane are formed by Haber-Weiss and Fenton reactions. Haber-Weiss reaction [28];

－→ ･OH + OH－ + O2 Fenton reaction [29];

H2O2 + O2

Cu+ ＋ H2O2 → ･OH ＋ OH－+ Cu2+

Furthermore, new ROS productions occur by Fenton-like type. L=Ligand

LCu (Ⅱ) + H2O2 → LCu (Ⅰ) + ･OOH + H＋

LCu (Ⅰ)＋H2O2 → LCu (Ⅱ)＋･OH + OH― Lipid peroxidation and the radical are accumulated, in which cell membrane oxidation and the decreasing function induce. Superoxide anion radical O2―thathydroxyl radical･OH induce ROS production, the － induce SOD production by hydrogen peroxide H2O2. Especially, and high toxic hydroxyl radical･OH can damage a range of cellular macromolecules, including causing mutations in DNA [30]. As above-mentioned, the bactericidal processes of bacteriolysis of the S.aureus and E.coli cell walls by Cu2+ ions, and also the antibacterial activities of cell membrane and cytoplasm are shown in Table 3.

other O2

Cu2+ionCell wallCell membraneCytoplasm
solution
Cu2+S.aureus cell wall･Cu-ammine
complex
and
Cu-protein
formations
Cu+, Cu2+
O ―
2
H O
2 2
･OH
OH－
･OOH
･OH･ formed by
Haber-
Weiss/Fenton
reactions
･Accumulation
of O ― and H O
2 2 2		･Cu-complex
and
Cu-3N-O
complex
formations
Cu2+
･OH
OH－
(O －）
2
H O
2 2
･DNA/RNA
synthesis
inhibition
Substitution
of Cu2+ ions
into DNA
hydrogen
bond base
pairs
PGN layer cell wall
Cu2+, Cu+
･Bacteriolysis of PGN cell wall by TG,TP synthesis inhibitions
and activations of S. Aureus PGN autolysins
･Cu complex of glycan saccharide chain and Cu peptide complex
formations
･Reactive oxygen species O ―,H O
2 2 2
Cu2+E.coli cell wall
Lipopolysaccharide
(LPS)		Outer
membrane(OM)
Lipoprotein(LPT)		Periplasmic
space(PS)
Core polysaccharide
Phosphorus lipid,		OmpF, A,C,
Porin, Protein		PGN layer
Miscible protein
Cu2+,Cu+
･Variation of
Charge properties
･Cu ion induced
increase in
permeability
･ O －, Cu+
2		Cu2+, Cu+
･Cu ion binding
proteins
･Damages of
outer membrane
structure due to
the degradable
enzymes of
lipoprotein at N-,
C-terminals and
the activation of
PGN hydrolases
･Cu+, H O
2 2		Cu2+, Cu+
･Cu2+ ions
accumulation
･Inhibitions of
PGN elongation by
the deletions of
PGN-TP
Enzyme and
PGN autolysins
･Cu+, HO･, H O
2 2

Table 3: Bactericidal action processes of Cu2+ solutions within the cell wall ／the cellmembrane ／the cytoplasm against S.aureus a

Conclusions

1. From the result of antibacterial activities of Cu2+ ion solution by the two-fold broth dilution medium method, for bacteriostasis MIC=50mg/L above was obtained in Cu2+ concentration range of 0.10～50 mg/L against E.coli. The other, for bactericide action MIC=625 mg/L and MBC=1250 mg/L were obtained in Cu2+ concentration range of 9.8～5,000 mg/L against S.aureus.

2. Bacteriolysis of S.aureus PGN cell wall by Cu2+ ions is caused for the inhibition of PGN elongation due to damages of PGN synthetic TG/TP and activation of PGN autolysins. The other, bacteriolysis of E.coli outer membrane cell wall by Cu2+ ions is attributed to the destruction of outer membrane structure and to the inhibition of PGN elongation due to the damage of PGN biosynthesis TP and the activation of PGN autolysins. 3. By the penetration of copper ions into bacterial cell －, H+, H2O2, ONOO－ occurs. The other, in E.coli cell wall, the productions of O2 wall, productions of O2 －, H+ in outer membrane, and H2O2, OH－, ･OH in periplasmic space occur. These ROS and H2O2 damage the cell membrane and the DNA molecules by oxidase stress.

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@article{ishida2017,
  title   = {Mechanism of Antibacterial Activities of Cu (â…¡) Ions against Staphylococcus aureus and Escherichia coli on the Ground of Results Obtained from Dilution Medium Method},
  author  = {Ishida T},
  journal = {Virology & Immunology Journal},
  year    = {2017},
  volume  = {1},
  number  = {3},
  doi     = {10.23880/vij-16000117}
}
Ishida T (2017). Mechanism of Antibacterial Activities of Cu (â…¡) Ions against Staphylococcus aureus and Escherichia coli on the Ground of Results Obtained from Dilution Medium Method. Virology & Immunology Journal, 1(3). https://doi.org/10.23880/vij-16000117
TY  - JOUR
TI  - Mechanism of Antibacterial Activities of Cu (â…¡) Ions against Staphylococcus aureus and Escherichia coli on the Ground of Results Obtained from Dilution Medium Method
AU  - Ishida T
JO  - Virology & Immunology Journal
PY  - 2017
VL  - 1
IS  - 3
DO  - 10.23880/vij-16000117
ER  -