https://en.wikipedia.org/wiki/Hydrogen_peroxide
http://jac.oxfordjournals.org/content/67/7/1589.full
http://jac.oxfordjournals.org/content/67/7/1589.full
Use of
hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal
action
+Author
Affiliations
Abstract
Hydrogen peroxide is
extensively used as a biocide, particularly in applications where its
decomposition into non-toxic by-products is important. Although increasing
information on the biocidal efficacy of hydrogen peroxide is available, there
is still little understanding of its biocidal mechanisms of action. This review
aims to combine past and novel evidence of interactions between hydrogen
peroxide and the microbial cell and its components, while reflecting on
alternative applications that make use of gaseous hydrogen peroxide. It is
currently believed that the Fenton reaction leading to the production of free
hydroxyl radicals is the basis of hydrogen peroxide action and evidence exists
for this reaction leading to oxidation of DNA, proteins and membrane
lipids in vivo. Investigations of DNA oxidation suggest that
the oxidizing radical is the ferryl radical formed from DNA-associated iron,
not hydroxyl. Investigations of protein oxidation suggest that selective
oxidation of certain proteins might occur, and that vapour-phase hydrogen
peroxide is a more potent oxidizer of protein than liquid-phase hydrogen
peroxide. Few studies have investigated membrane damage by hydrogen peroxide,
though it is suggested that this is important for the biocidal mechanism. No
studies have investigated damage to microbial cell components under conditions
commonly used for sterilization. Despite extensive studies of hydrogen peroxide
toxicity, the mechanism of its action as a biocide requires further
investigation.
oxidizing agentsdisinfectiontoxicitymechanism of actionPrevious SectionNext
Section
Introduction
The interest in
environmentally friendly, non-toxic and degradable yet potent biocides has
never been so high. Oxidizing agents, notably hydrogen peroxide (H2O2), are increasingly used
in a number of medical, food and industrial applications but also in
environmental ones such as water treatment. In the medical arena, oxidizing
agents are particularly useful for hard surface disinfection and the high-level
disinfection of medical devices. Their main advantages are their broad-spectrum
activity, which includes efficacy against bacterial endospores, their lack of
environmental toxicity following their complete degradation, and the fact that,
with imaginative formulation, their surface corrosiveness and smell (for
peracetic acid-based products) have been greatly reduced. H2O2 is particularly
interesting for its application in liquid but also vaporized form for
antisepsis and for the disinfection of surfaces and medical devices and for
room fumigation (the so-called deep clean).
H2O2 was discovered by
Louis Thénard in 18181 and its use as a
disinfectant first proposed by B. W. Richardson in 1891.2 It is now in
widespread use as a biocide, particularly in applications where its decomposition
into non-toxic by-products (water and oxygen) is important. For example, 3%–6%
(v/v) peroxide in water is widely used as an antiseptic (in particular on
wounds) and general surface disinfectant. Commercial dental disinfectant
formulations such as Dentasept® (Muller Dental) and Oxigenal (Kavo) use 1%
(294 mM) and 0.4% (118 mM) H2O2 as an active ingredient, respectively.3 Many contact lens
disinfectant solutions use 3% (882 mM) H2O2 as an active
ingredient or preservative, including Concerto (Essilor), Oxysept® 1 Step (Abbott),
Multi™ (Sauflon) and AOSept® 1-step (Ciba Vision).4
The relative safety of H2O2 solutions has meant
that it had also found extensive use in the food industry. Sapers and Sites5 discuss a commercial
post-harvest wash (Biosafe®) that uses H2O2 as an active
ingredient at an in-use concentration of between 0.27% (79 mM) and 0.54% (159
mM) and a surface disinfectant (Sanosil-25) with an in-use concentration of
0.24% (71 mM) H2O2. They also demonstrated the efficacy of 1% (294
mM) H2O2as a wash to decontaminate
apples.
Nikkhah et al.6 state that ‘hydrogen
peroxide is the most commonly used packing sterilant in aseptic processing
systems’. Typically it is used at very high concentrations (35%, 10.3 M) and
often in combination with heat.7 In contrast, H2O2 gas (often referred
to as vaporized or gaseous H2O2) is typically used at much lower concentrations
and temperatures; indeed it has been suggested that the biocidal activity of
gaseous peroxide is distinct from that of liquid peroxide.8,9
There is remarkably little
literature discussing the exact mechanism(s) of the biocidal action of H2O2. As suggested in general
reviews on the mechanisms of action of biocides, H2O2 is considered an
oxidizing agent reactive with the biomolecules (proteins, lipids, nucleic
acids, etc.) that make up cellular and viral structure/function.10,11 This situation is
complicated by the importance of H2O2 as a physiological
source of reactive oxygen species (ROS) in respiring cells and as a component
of the human innate immune system. The majority of studies investigating the
toxic mechanism of H2O2 therefore consider it as a source of
oxidative stress in the cell to model chronic oxidative damage to cells or to
investigate the various killing mechanisms of leucocytes.12
This review aims to
provide a critical overview of the body of knowledge on the toxic mechanisms of
H2O2 and, importantly, to
examine the relevance of various frequently cited studies to the understanding
of the biocidal mechanism of H2O2 at typical in-use concentrations, in both
the liquid and gaseous phases.
Mechanism of cytotoxicity of H2O2
Radical
formation and Fenton reaction
The mechanism of cytotoxic
activity is generally reported to be based on the production of highly reactive
hydroxyl radicals from the interaction of the superoxide (O2•−) radical and H2O2, a reaction first
proposed by Haber and Weiss13 (eq. 1):1
Further, it is believed
that the production of extremely short-lived hydroxyl radicals within the cell
by the Haber–Weiss cycle is catalysed in vivo by the
presence of transition metal ions (particularly iron-II) according to Fenton
chemistry14 (eq. 2):2
It is known that, in vitro, the hydroxyl radical and other oxygenated
species can act as potent oxidizing agents, reacting with lipids, proteins and
nucleic acids.15 It is easy to
propose that such reactions can account for the antimicrobial effects of H2O2 and a number of
scientific publications have used such an explanation to describe the action of
the oxidizing agents.
Evidence
for Fenton-like chemistry in biocidal activity of H2O2
Evidence for this
mechanism in the action of H2O2 on bacterial cells was provided by
Repine et al.16 They demonstrated that
growingStaphylococcus aureus overnight in Bacto nutrient
broth containing increasing concentrations of iron resulted in an increase in
intracellular iron content. Incubation of harvested cells at 37°C in Hanks’
balanced salt solution containing a range of H2O2 concentrations
showed that those cells with an elevated iron concentration had greater
susceptibility to H2O2, as measured by a decrease in the H2O2 concentration
required to kill 50% of cells after a 60 min exposure. An increase in iron
concentration in the growth medium was shown to have no effect on the growth
rate or viability of S. aureus or on
catalase or peroxidase activity. Repine et al.16also showed that the addition
of thiourea, dimethyl thiourea, sodium benzoate and dimethyl sulphoxide
inhibited the toxic effects of H2O2 in proportion to the effectiveness of the
substances as hydroxyl scavengers. Again, the addition of these substances had
no effect on viability, nor were they found to directly react with H2O2. Further evidence was
found by Mello Filho et al.,17 who showed that the
potent iron chelator 1,10-phenoanthroline (1,10-phen) could penetrate cultured
mouse cells and protect them against killing by H2O2. The chelator alone had
no effect on cell viability.
Indirect
evidence of DNA damage
Imlay and Linn18 exposed Escherichia coli K12 to varying concentrations of
H2O2 for 15 min at 37°C
in K medium. They observed that cells were more susceptible to low (<3 mM)
concentrations of H2O2 than to intermediate (5–20 mM)
concentrations. At >20 mM H2O2, survival was inversely proportional to
concentration. This response is shown in Figure 1. A slight dip in the
surviving fraction of the culture can be seen at concentrations <3 mM. This
effect was found to be reproducible and was greatly magnified in DNA
repair-deficient and anoxically grown strains; these were particularly
sensitive to H2O2 at low concentrations but not especially
susceptible to higher concentrations when compared with aerobically grown
wild-type cells. Cells starved by incubation in M90 salts for 80 min before
exposure to H2O2 were not killed by low concentrations of H2O2. Comparisons of the
kinetics of killing of an exonuclease-II-deficient strain at various H2O2 concentrations were
also made. Total kill by both lower and higher H2O2 concentrations was
found to be time-dependent (Figure 2). They postulated that
killing of E. coli cells by H2O2occurs according to two
distinct modes, with mode-1 killing occurring at low concentrations due to DNA
damage and mode-2 killing occurring at higher concentrations due to damage to
other target(s).
View larger version:
Figure 1.
Chart
showing the log10surviving fraction of wild-type E. coli K12 culture after 15 min of exposure to
various H2O2concentrations. Adapted
from Imlay and Linn18 with permission from
the American Society for Microbiology.
View larger version:
Figure 2.
Chart
showing change in the surviving fraction with time of exposure to 1.25 mM H2O2 (mode-1 killing) and
25 mM H2O2 (mode-2 killing).
Adapted from Imlay and Linn18 with permission from
the American Society for Microbiology.
Similar results were
obtained by Brandi et al.,19 who also observed
the bimodal killing pattern seen by Imlay and Linn18 after
challenging E. coli in M9 salts with
various H2O2 concentrations for
15 min. In addition, it was found that mode-2 killing was markedly reduced in
anoxic conditions, whereas no effect was seen on mode-1 killing.
Brandi et al.19 also compared the
effect of the hydroxyl scavenger thiourea on killing by 2.5 or 25 mM H2O2. Thiourea at 35 mM was
found to markedly reduce killing by 25 mM H2O2 (hypothesized to be
due to the mode-2 mechanism) whilst the same concentration had no effect on
killing by 2.5 mM H2O2 (hypothesized to be due to the mode-1
mechanism). An obvious criticism of this work is that thiourea, a potent
reducing agent, is capable of reacting directly with H2O2. This is a first-order reaction
with respect to H2O2 concentration, so it is possible that the
thiourea simply reduces the higher concentration of H2O2 without scavenging
hydroxyl radicals. Unlike the earlier work of Repine et al.,16 it was not checked
that this direct reaction did not occur in their test system, nor were
alternative OH• scavengers tested.
Brandi et al.19 concluded that
mode-2 killing was dependent on the presence of oxygen and hydroxyl radicals,
and suggested that this mechanism is indeed due to the Fenton chemistry
previously outlined, whilst the mode-1 killing was not dependent on oxygen and
hydroxyl radicals.
A study by Macomber et al.20 using copper
export-deficient strains of E. coli grown
in a copper-supplemented medium showed that these strains accumulated copper
within the cell, but that this increase actually inhibited both killing and
mutagenesis in a DNA repair-deficient strain by millimolar concentrations of H2O2. Though they could find
no definitive explanation for the inhibitory effect of the copper, their work
shows that mode-1 and mode-2 killing due to DNA damage is not mediated by
copper.
In vitro
investigations of DNA damage
Due to the relevance of
mode-1 killing as a general model of oxidative DNA damage under aerobic
conditions and its usefulness in elucidating the cellular mechanisms of
prevention and repair of such damage, much subsequent work has focused on the
mode-1 mechanisms of DNA damage. Imlay et al.21 developed an in vitro model capable of producing the same
pattern of damage to purified DNA as that observed in the process of killing of
bacteria. This model consisted of phage PM2 DNA incubated with ferrous sulphate
and various concentrations of ethanol and H2O2. This system produced
single-strand breaks in the purified DNA. The production of breaks was reduced
by approximately half by the addition of micromolar concentrations of ethanol,
but was not further decreased by the addition of up to 10 mM ethanol. Addition
of a range of H2O2 concentrations to the system containing 10
mM ethanol produced a DNA nick dose response similar to that seen in mode-1
killing of E. coli; the highest number of
nicks was produced by 50 μM H2O2, and this was reduced by
half and remained approximately constant on addition of 1–10 mM H2O2. As ethanol is a potent
scavenger of hydroxyl radicals, it was concluded that the mode-1 killing of
bacterial cells due to DNA damage by H2O2 is not dependent on
the production of free hydroxyl radicals, and is more likely to be due to the
production of ferryl radical intermediates from DNA complexation.
Further work on Imlay and
Linn's in vitro model by Luo et al.22 found that DNA nicking
was maximal at 50 μM H2O2, dropping to one-third
maximal at 3 mM and remaining roughly constant between 3 and 50 mM. Addition of
17 μM ethanol to the model
reduced nicking by 30%–50% at all H2O2concentrations. Increasing
the ethanol concentration to 10 mM reduced nicking by a further 50% at H2O2 concentrations
<100 μM, but had no effect at
higher peroxide concentrations. Increasing the ethanol concentration to 100 mM
caused further inhibition at H2O2 concentrations <3 mM, but again had no
effect at higher concentrations. These findings are summarized in Table 1. Luo et al.22 therefore concluded
that there are at least three chemically distinct classes of oxidant species
produced by Fenton-type reactions in the presence of iron: type I are sensitive
to H2O2but moderately resistant
to ethanol; type II are resistant to both H2O2 and ethanol; and
type III are sensitive to H2O2 and ethanol.
Ethanol concentration | <100 μM H2O2 | 0.1–3 mM H2O2 | 3–50 mM H2O2 |
---|---|---|---|
No ethanol | maximal nicking | 1/3× maximal nicking | 1/3× maximal nicking |
17 μM | reduced nicking | reduced nicking | reduced nicking |
10 mM | further reduced nicking | further reduced nicking | no further reduction in nicking |
100 mM | further reduced nicking | no further reduction in nicking | no further reduction in nicking |
View this table:
Table 1.
Effects of
increasing H2O2and ethanol concentration on DNA nicking in vitro; summarized from Luo et al.22
Evidence
for different types of DNA oxidation reactions
Luo et al.22 went on to
investigate the effects of the iron chelators 1,10-phen and 2,2′-dipyridyl
(2,2′-dipy) both on E. coli killing
and on DNA nicking in the in vitro model.
Both chelators blocked mode-1 killing, whilst 2,2′-dipy had no effect on mode-2
killing, and 1,10-phen substantially enhanced it. Both chelators also blocked
DNA nicking at low H2O2concentrations but not at higher concentrations;
the peak of DNA nicking activity at 50 μM H2O2 was completely
eliminated by the addition of either chelating agent plus 100 mM ethanol, while
nicking was constant at around 50% of the maximum between 50 μM and 50 mM H2O2 with 2,2′-dipy and
100 mM ethanol, and was enhanced 6-fold by the addition of 1,10-phen between 50
μM and 50 mM H2O2. As 2,2′-dipy chelates
unbound Fe2+ and remains in
solution, whilst 1,10-phen chelates unbound Fe2+ and then
intercalates into the DNA backbone, they proposed the following model of
oxidant damage. Type I oxidants are formed by Fe2+ ions associated but
not bound to DNA—suggesting that these could exist in a ‘cationic cloud
surrounding the polyanionic DNA helix’ (Luo et al.22). Such oxidants are
accessible to H2O2 quenching, but would require higher
concentrations of ethanol to effectively quench them due to high localized
concentrations within the ‘cationic cloud’. These oxidants are responsible for
mode-1 killing. Type II oxidants are formed by Fe2+ more tightly
associated with DNA, and once formed they are not accessible to H2O2 or ethanol
quenching, and are responsible for mode-2 killing. Type III oxidants are
produced by free Fe2+ ions in solution, they are easily available
to H2O2 and ethanol to
quench, and due to their short half-life are unlikely to be involved in killing
due to DNA damage in vivo. The action of the
chelating agents can be explained thus: 2,2′-dipy removes the ‘cationic cloud’
and causes the formation of type III rather than type I oxidants, inhibiting
mode-1 killing; 1,10-phen removes the ‘cationic cloud’ and possibly free Fe2+ ions and
intercalates them into the DNA backbone and causes the formation of type II
rather than type I or type II oxidants, enhancing mode-2 killing and inhibiting
mode-1 killing. These findings are summarized in
Table 2.
Property | Type I | Type II | Type III |
---|---|---|---|
Resistant to H2O2? | no | yes | no |
Resistant to ethanol? | moderately | yes | no |
Effect of 1,10-phen | decreased | increased | possibly decreased |
Effect of 2,2′-dipy | decreased | none | increased |
Position | cationic cloud | DNA backbone | free in solution |
Killing mode | mode-1 | mode-2 | none |
View this table:
Table 2.
Studies by Henle and Linn23 suggest that the
type I and II oxidants have different preferred cleavage sequences on the DNA
molecule, with type I preferentially cleaving the sequences RTGR, TATTY and CTTR and type II preferentially cleaving the
sequence NGGG (where the underlined bases show the cleavage
site). They suggested that this selectivity could be due to sites of iron
localization, or that such sequences act as sinks for radical electrons formed
elsewhere on the DNA chain.
Direct
evidence of DNA damage in vivo
None of the studies
described above directly measured DNA damage in vivo. DNA
repair-deficient strains were found to be more susceptible to killing, and so
it was concluded that DNA damage was the cause of death, but it could be
suggested that this does not logically follow. One can equally easily imagine
that lethal DNA damage in fact only occurs in repair-deficient strains and this
acts in addition to some other damage to increase the bactericidal effect seen
with wild-type strains. Whilst the in vitro studies
performed using Imlay and Linn's model add support to the hypothesis that DNA
damage is the main cause of the bactericidal effect of low concentrations of H2O2, they do not provide
proof that such damage also occurs in vivo. Studies
that directly measure DNA damage caused by H2O2 have been performed
using several different methods to estimate different types of DNA damage.
Ananthaswamy and
Eisenstark24 exposed E. coli strain W3110 to 10 mM H2O2 in phosphate buffer
for 10 min at 25°C then measured the number of single-strand breaks formed
using alkaline sucrose gradient sedimentation. They found that the treatment
produced 153 single-strand breaks per genome, of which all but 74 could be
repaired by further incubating the culture for 15 min in phosphate buffer at
25°C, and all but 14 could be repaired by incubating the culture for 40 min in
M9 medium at 37°C.
Hagensee and Moses25 exposed E. coli strain W3110 to 117 mM H2O2 in phosphate buffer,
pH 7.4, for 10 min at 37°C then measured the number of single-strand breaks
formed using alkaline gradient centrifugation. They found that the treatment
produced 482 single-strand breaks per genome, and all but 18 could be repaired
by incubating the culture for 4 h in M9 medium at 37°C.
Rohwer and Azam26 exposed
exponential-phase cultures of E. coli strain
K37 and the archaeon Haloferax volcanii to
0.2% (59 mM) H2O2 in Luria–Bertani (LB) broth or H. volcanii medium, respectively, for 30 min at
room temperature. The treated cultures were then analysed using the terminal
deoxyribonucleotide transferase-mediated dUTP nick end labelling (TUNEL) method
to label 3′-OH DNA ends and flow cytometry. They found that 97.4% of the H2O2-treated E. coli cells were TUNEL positive (i.e. had 3′-OH
ends) compared with <1% of control cells. Similarly, 84.3% of treated H. volcanii cells were TUNEL positive compared
with 9.6% of control cells. This effect could be reduced by pre-treating the
cultures with protein synthesis inhibitors: chloramphenicol pre-treatment
of E. colireduced the percentage of TUNEL-positive cells
after H2O2 exposure to 7.8%,
whilst diphtheria toxin pre-treatment reduced the percentage of
TUNEL-positive H. volcanii cells to 31.4%.
Exposing the E. coli culture to H2O2 for a longer time
(60 min, compared with 30 min) increased fluorescence in TUNEL-positive cells,
as did treatment with an increased concentration of H2O2 [0.4% (118 mM),
compared with 0.2%]. Stationary-phase E. coli cultures
exposed to 0.4% H2O2 for 30 min did not demonstrate any detectable
breaks. The authors therefore concluded that H2O2 exposure results in
oxidation of DNA bases and that these oxidized bases are recognized and excised
by DNA repair mechanisms, resulting in single-strand breaks in the DNA
molecule. The amount of breaks thus produced increases with exposure time and
concentration of H2O2.
Fernández et al.27 exposed both
exponential- and stationary-phase E. colistrain TG1
cultures to 10 mM H2O2 in LB broth for 10 min at room
temperature. DNA damage was then assessed using a diffusion assay, and 100% of
nucleoids in both stationary and exponential phases were found to show
‘extensively fragmented DNA’, compared with 0.4% and 37.6% of untreated exponential-
and stationary-phase cultures, respectively. They therefore concluded that
exposure to H2O2 causes substantially more damage to DNA
than can be assessed using the TUNEL method—i.e. the TUNEL method can detect
only the formation of 3′-OH ends in single-strand breaks, and these are not the
only lesions that occur.
Summary of
evidence of DNA damage by H2O2
Whilst the studies
described here give a very complete model of the genotoxic action of low
concentrations of H2O2 on bacterial cells, one should use caution
in applying these findings to the bactericidal mechanism of H2O2 as a disinfectant.
None of these studies simultaneously measured reduction in cell count and
formation of DNA damage for a range of H2O2 concentrations
and/or exposure times in order for a correlation between cells killed and DNA
damage to be calculated. Variations in strains and species used, treatment
conditions and exposure times also make comparison of the studies difficult. In
particular, the media used to treat cultures varied greatly between the
studies, from simple phosphate buffer to complex media such as LB broth. It has
been shown that the presence of other substances can have a significant effect
on the efficacy of bactericidal action of H2O2, both positive and
negative. For example, Berglin et al.28 showed that the
presence of 0.1 mM cysteine in the growth medium increases sensitivity ofE. coli strain K12 100-fold to 0.1 mM H2O2, so conclusions about the
bactericidal mechanism need to take media composition into account.
Most crucially, the
majority of the studies described used low concentrations of H2O2 and long exposure
times—typically 50 μM to 2.5 mM H2O2 with >15 min of
exposure. In contrast, the typical concentration of surface sterilant solution
of H2O2 is 3%, equivalent to
882 mM. The literature on DNA oxidation by low concentrations of H2O2 is therefore of
questionable significance to the use of H2O2 as a surface
disinfectant. As mode-2 killing due to DNA damage is hypothesized to be reliant
on Fe2+ions closely associated
with the DNA molecule and the presence of cellular reductants to drive the
Fenton cycle, it is possible that the DNA damage dose–response to H2O2 will reach a maximum
at some concentration of H2O2, and that damage to other cellular components
will start to become more important at higher concentrations. The mechanisms of
H2O2damage to the other major
macromolecular targets within the bacterial cell, namely protein and lipids,
are not so well studied as DNA damage.
Interactions
of H2O2 with
proteins and amino acids
Unlike DNA damage,
where in vitro work shows that H2O2 alone is unreactive
with DNA, a mechanism exists for the non-radical-based reaction of H2O2 with proteins even
in the absence of metal ions. Luo et al.29 and Ashby and Nagy30 discussed in detail
the kinetics and mechanism of the reaction of H2O2 with cysteine in the
absence of metal ions. Kim et al.31 developed a method
based on the selective and competitive reaction of H2O2 and
biotin-conjugated iodoacetamide with cysteine residues exhibiting low pKa to allow labelling of proteins containing
such residues. Using this method, they identified several proteins present in various
mammalian cells lines that are preferentially oxidized by H2O2. Finnegan et al.9 have also
demonstrated oxidation of cysteine, methionine, lysine, histidine and glycine on
exposure of 100 mM amino acid to 100 mM H2O2 in the absence of
added metal ions.
Returning to the
metal-ion-catalysed production of hydroxyl radicals, oxidation of several amino
acid residues has been shown to occur by this reaction; Dean et al.32 summarized the
various products of radical-mediated oxidation of amino acids. The oxidation of
protein amino acids can result in a range of modifications, from total cleavage
of the protein backbone to subtle side-chain modification of individual
residues. The production of carbonyl residues is often used as an indicator of
protein oxidation, as it is the outcome of many oxidative modifications and is
easily quantified. The effect of H2O2 treatment on the
amount of protein carbonyls has been assessed by several groups, though, as for
DNA damage, H2O2 treatment was generally considered as a
source of oxidative stress rather than as a biocide.
Tamarit et al.33 grew E. coli K12 strain ECL1 anaerobically and
challenged these cultures with 2 mM H2O2 for up to 45 min,
following which 1 mL samples of the cultures were taken and crude protein
extracts prepared. The protein extracts were derivatized with dinitrophenyl
hydrazine (DNPH) and separated by one-dimensional SDS-PAGE. Western blot
immunoassay was used to detect DNPH-derivatized carbonyl groups on the protein
bands, and bands of interest were identified using Edman degradation. It was
found that H2O2 stress caused a 30% reduction in cell
viability (as previously discussed, mortality under these conditions has been
suggested to be mostly due to mode-1 killing) and a 3-fold increase in protein
carbonyl content of crude extract. Protein bands exhibited widely varying
increases in carbonyl content—several proteins, such as alcohol dehydrogenase
E, enolase, DNA K, EF-G and an outer membrane protein A, showed a substantial
increase in carbonyl content, whilst two major protein bands (EF-Tu and outer
membrane protein C) were not oxidized at all. This same pattern was seen with
cells grown under aerobic conditions, though the increase in carbonyl content
here was not so dramatic (50% compared with 300%) due to the activation of
oxidative stress response systems.
Cabiscol et al.34 went on to repeat
this work with the yeast Saccharomyces cerevisiae treated
with 5 mM H2O2 for 45 min, and again a selective pattern
of protein oxidation was observed.
Whilst the work of
Tamarit et al.33 and Cabiscol et al.34 was performed using
low concentrations of H2O2 more usually associated with mode-1
killing, the exposure time of 45 min used was far longer than the normal
exposure time tested for a biocide. In other words, it is possible that the selective
oxidation of proteins by low concentrations of H2O2 over long exposure
times could be reproduced by high concentrations of H2O2 over short time
scales. The action of 3% H2O2 as a surface disinfectant might therefore
be far more selective, in terms of those proteins most damaged, than often
considered.
At this stage it is
appropriate to reflect on the different effects that can be observed with H2O2 when tested diluted
in water, in formulation with other chemicals or, notably, when in gas form.
Finnegan et al.9 observed important
differences in the interaction of vaporized (gaseous) and liquid H2O2 against amino acids.
Vaporized H2O2 (2 mg/L for 10 min, when tested under true
gas, non-condensed conditions) was shown not to be able to oxidize amino acids
(100 mM) whereas liquid H2O2 was shown to oxidize cysteine, methionine,
lysine, histidine and glycine at various H2O2/amino acid ratios.
However, both liquid (12 mg/L) and vaporized H2O2 (2 mg/L for 10 min)
completely degraded BSA and aldolase. Others have reported potential
cross-linking effects on protein exposure to liquid peroxide but protein
degradation (into smaller peptides) on exposure to gaseous peroxide.35,36 It is clear that
liquid and gaseous H2O2 interact differently with macromolecules,
which may explain their differences in biocidal efficacy.
Interactions
of H2O2 with
bacterial cell membranes and lipids
Studies of the effect of H2O2 on bacterial cell
membranes are also limited. Whilst much work has been performed using H2O2 as a source of ROS
to simulate the effects of oxidation during ageing on mammalian cells, a
literature search using PubMed found only three studies investigating the
effects of H2O2 on the membrane of any bacteria.
Brandi et al.37 exposedE. coli cells to mode-1 and mode-2 concentrations
of H2O2 and studied effects
on cell morphology and the cell membrane. They found that low concentrations
(1.75 mM), producing mode-1 effects, caused extensive cell filamentation, but
that this change in morphology did not occur at higher (17.5 mM) H2O2 concentrations;
instead a large decrease in cell volume was observed.
Brandi et al.37 also found that loss
of intracellular contents, as measured by lactate dehydrogenase activity in
culture medium, occurred at a low rate initially under mode-1 killing
conditions, and ceased after 150 min. In contrast, the higher concentration of
H2O2 produced a much
larger increase in lactate dehydrogenase activity in the culture medium. They
hypothesized that the reduction in cell volume seen during mode-2 killing was
due to cell membrane damage and loss of intracellular material, and suggested
that this was the major component of mode-2 killing.
Baatout et al.38 exposed Ralstonia metallidurans, E. coli, Shewanella oneidensis and Deinococcus radiodurans cultures to concentrations
of H2O2 up to 880 mM (the
only study to investigate the typical 3% H2O2used in disinfectant
solutions) for 1 h then measured various indicators of cell physiology. They
found that cell membrane permeability, as measured by propidium iodide uptake,
was markedly increased in all strains at H2O2concentrations >13.25
mM.
Finally, Peterson et al.39 measured the release
of organic compounds from the cyanobacterium Aphanizomenon flos-aquae after
exposure to various water treatments, including H2O2. They found a substantial
increase in the release of dissolved organic compounds and the odour compound
geosmin with increasing H2O2 concentration up to 0.025% (equivalent to
0.73 mM), and that cell membrane damage, as measured by potassium leakage, also
increased with peroxide concentration up to 0.01% (equivalent to 0.29 mM).
All three studies showed
some degree of cell membrane damage due to exposure to H2O2, and Brandi et al.37 suggested that such
damage may be a major component of mode-2 killing of E. coli.
Vaporized H2O2 as a
sterilant
The use of vaporized (or
gaseous) H2O2 as a sterilant was
pioneered in the packaging industry by Wang and Toledo in the late 1980s.40 Its use in
preference to a liquid solution of 35% (equivalent to 880 mM) H2O2 was initially
investigated in order to avoid residual traces being retained on packaging;
however, it also offers the advantage over liquid in that large volumes and
devices that might be damaged by exposure to water can be easily sterilized.
Other advantages over alternative vapour-phase methods (e.g. based on ozone or
peracetic acid) are low toxicity and spontaneous breakdown into completely
harmless by-products.40
Since Wang and Toledo's
initial work, commercial vapour-phase H2O2treatments have been
developed and their efficacy investigated in several applications, including
decontamination of laboratory and medical equipment, hospital wards and
pharmaceutical manufacturing facilities. They have been shown to be efficacious
against a wide range of organisms, including those producing endospores,41,42 Gram-positive and Gram-negative
vegetative cells,43,44 DNA and RNA viruses45–47 and fungi.44,48,49 These systems vary
in their use of H2O2, ranging from pure gas-based processes (often
referred to as ‘dry’), condensed peroxide (formed from a saturated gas,
referred to as ‘wet’) and liquid misting systems (for liquid peroxide
distribution within an area50). Antimicrobial efficacy,
surface compatibility and safety aspects can vary between these systems.
Despite the increasing use
of such decontamination methods and the growing body of literature detailing
the validation of these methods for use in various applications, it appears
that little work has been done on understanding the mechanism(s) of biocidal
activity of the vapour form of H2O2. Indeed, one early study of a vapour system to
sterilize centrifuges performed by Klapes and Vesley51 concluded ‘The
application of VPHP [vapour phase H2O2] as a potential sterilant
is still clearly in its infancy: definitive knowledge of the mechanism(s) of
cidal action, and the factors which influence it, is lacking’, whilst a study
of the use of H2O2 vapour to deactivate Mycobacterium tuberculosis performed by Hall et al.52 stated in the
conclusion ‘the exact mechanism of action of
HPV remains to be fully elucidated’.
The lack of investigation
into the vapour-phase H2O2 cidal mechanism appears more remarkable in
the light of a study performed by Fichet et al.,35 which showed that,
of several decontamination methods tested, gaseous H2O2 prevented the
exhibition of spongiform pathologies in hamsters following intercerebral
inoculation with steel wires contaminated with infectious brain matter and then
treated with the decontamination methods.
Despite this, Yan et al.53 reported little
effect with an H2O2 gas plasma system, but this may be
explained by the fact that such a system can be associated with condensed
(therefore liquid/gas) H2O2 in contrast to a non-condensed gas-based
process (as discussed by Fichet et al.35,36). A follow-up study
performed by Fichet et al.36 with a vacuum-based
sterilization process using non-condensed gas reproduced this destruction of
infectivity with gaseous H2O2 but not liquid H2O2. In vitrostudies showed unfolding and degradation of the
prion proteins by gaseous but not liquid H2O2.
These studies suggest that
the ability of H2O2 to degrade protein oxidatively is greatly
enhanced in the vapour phase compared with the liquid phase. This had also been
observed in studies with the neutralization of bacteria protein toxins, such as
those produced byClostridium botulinum and Bacillus anthracis (McDonnell8). Further evidence for
this was provided by Finnegan et al.,9 who showed that
vaporized H2O2 could completely degrade BSA and aldolase,
whilst liquid H2O2 had no effect on either. These results
suggest that there are subtle differences in the mechanisms of action of liquid
and gaseous H2O2, with potential impact on antimicrobial
efficacy. This may not only be the case with protein neutralization or prion
infectivity reduction. It has been known for some time that the organism most
resistant to liquid peroxide appears to be Bacillus subtilis (or Bacillus atrophaeus), in contrast to that for gaseous
peroxide, which is Geobacillus stearothermophilus.48Similarly, antimicrobial
efficacy against viruses can vary between condensed and non-condensed peroxide
disinfection processes, with the condensed (high peroxide concentration liquid)
systems potentially allowing viruses to be protected from the antimicrobial
effects of the liquid/gas.44,46
Conclusions
The two decades since
Imlay and Brandi's initial demonstration of the existence of the bimodal effect
have seen studies showing that the precise mechanism of genotoxicity of low H2O2 concentrations is
far subtler than might initially have been thought. Although H2O2 is obviously far too
simple a substance to exhibit innate selectivity of action, such is the
complexity of the cellular environment that it would nevertheless appear that H2O2 genotoxicity occurs
due to two distinct mechanisms, both of which cleave preferentially at
different sets of nucleotide sequences. H2O2might itself act as a sink
for the majority of radicals produced by Fenton chemistry in the target cell,
with only those radicals produced immediately proximate to the DNA chain able
to react and cause damage. Based on this observation, it is possible that, at
higher concentrations of H2O2, the amount of DNA-associated iron becomes the
limiting factor in the reaction between H2O2 and DNA, and thus
further increases in the H2O2concentration might not necessarily result in an
increase in the rate of DNA damage. Oxidation of proteins and lipids could thus
be more significant to the biocidal mechanism at higher concentrations of H2O2.
Compared with DNA damage,
oxidation of other bacterial cell components by H2O2 is far less studied,
though evidence exists of damage to both proteins and the cell membrane. A
number of studies have suggested that oxidation of bacterial proteins is also a
more selective process than typically reported, with specific proteins being
more or less vulnerable to oxidation.
Whilst the studies
described provide clues as to the biocidal mechanism of H2O2, there is a limit to how
much can be learned about this from studies designed to investigate the effect
of oxidative stress on a particular type of macromolecule. For instance, a
study designed only to investigate damage to DNA can tell us whether such
damage occurs, but will give no information as to how important this damage is
to the bactericidal effect; it is impossible to draw conclusions as to the
importance of the putative lesions to the lethal mechanism of H2O2 without a
simultaneous measurement of damage to all bacterial cell components, and a
correlation of this damage with a reduction in viable cell count. Such a study
has not been performed, and consequently our knowledge of the bactericidal
mechanism of H2O2 action at the molecular level must be
considered incomplete, especially with the high H2O2 concentrations and
short contact times representative of H2O2 biocidal
applications. Certainly, examination of the evidence appears to dispel the
model of free hydroxyl radical production as the main mechanism of H2O2 action at higher
concentrations.
Finally, it is apparent
that there is likely a qualitative difference between the biocidal mechanisms
of liquid-phase and gas-phase H2O2 that cannot be explained by current
models. Evidence suggests that H2O2 vapour can fragment protein without
Fenton-like reactions and the importance of this phenomenon needs to be
examined.
Despite extensive studies
of H2O2 toxicity, the
mechanism of its action as a biocide requires further investigation. This may
assist in the optimization of its antimicrobial effects for future
antimicrobial and neutralization applications.
Funding
This work was supported by Cardiff University
and Steris Ltd.
Transparency
declarations
G. M. is an employee of, and a minor stockholder
with, Steris Ltd. All other authors: none to declare.
·
© The Author 2012. Published by Oxford
University Press on behalf of the British Society for Antimicrobial
Chemotherapy. All rights reserved. For Permissions, please e-mail:
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