Peroxygen Compounds



Peroxygen Compounds


John Matta

Maruti Sinha

Kris Murphy

Suranjan Roychowdhury



Research efforts in chemical disinfection and sterilization research and development have taken many turns over the years. Recent advances and future guidelines seem clear: We will concentrate on chemicals that will be effective against microorganisms when highly diluted; will be low in toxicity to people, animals, aquatic life, etc; will not injure or have minimal impact on the environment; and will meet existing and developing regulatory requirements internationally. One such chemical, which will be considered in this chapter, was appraised many years ago by Dr. Samuel S. Wallian, in an address to the New York State Medical Society in 1892:


One can hardly refer to the medical journals without finding enthusiastic recommendations of it as a disinfectant of rare efficiency, an antiseptic of recognized merit and a germicide of decided potency…. It is also a reliable sporicide, and at the same time it is nontoxic and noncorrosive qualities possessed by few if any of the other sporicides yet brought to notice.

The chemical Dr. Wallian referred to was then known as “oxidized water”; we know it as hydrogen peroxide (HP), which is the most common example of a peroxygen compound. Peroxygen compounds contain the functional group R-O-O-R, where R stands for either hydrogens or an organic group; it is the -O-O- functional structure that defines peroxides. Peroxide compounds are powerful oxidizing agents and are often thought of in terms of their bleaching power. But the peroxo group is also very reactive itself and in forming associated radicals and in attacking various organic compounds such as proteins, lipids, and nucleic acids. These effects can accumulate in potent antimicrobial activity. This chapter will address the use of peroxygen compounds, mainly HP and peracetic acid (PAA), in disinfection and sterilization. This chemistry has become very important because of the stability and ease of use of these compounds. Other similar chemicals are considered elsewhere in this book, such as chlorine dioxide (see chapter 27) and ozone (see chapter 33).


HYDROGEN PEROXIDE

The HP has had a rocky road in its acceptance as a disinfectant; first popular, then unpopular, and more recently finding special application to serve functions of great value. Important early uses, in the preantibiotic world, were for wounds and for treating diphtheria. Eventually, its use expanded to general surface disinfection, treatment of skin infections, cleaning, air fumigation or disinfection, sterilization, and as an oral rinse or for direct use in the eye (at low concentrations). It is considered so safe under certain conditions that it was approved for use in foods in many countries.1 One of the major applications in both liquid and gas form is in sterilizing containers for aseptically preserved foods like fresh milk and fruit juices. The HP can be easily destroyed by heat or by enzymes such as catalase and peroxidase to give the innocuous end products, oxygen and water.

The use of HP as a disinfectant can be traced back to the late 1800s.2 First reported in the early 1800s by Louis-Jacques Thenard,3 it was used primarily as a skin antiseptic. As industrial production and uses of HP developed through the 1800s, the wider application of peroxide in clinical and therapeutic uses was proposed by B. W. Richardson in 1891.4 Although the germ theory of disease was still at a very early stage of understanding, it was noted that antimicrobial methods such as boiling water and the use of certain types of chemicals had a benefit in reducing the spread of disease or promoting healing. The antiseptic effects of HP solutions used on would infections is credited to Richardson in 1856.1 Even at this early time in disinfection, there were reports of using peroxide as an aerosol or gas to refresh the air by DeSondalo (1842), in meeting places and mines.2 The combination of
developments in peroxide use for disinfection along with improved methods for bulk manufacture of HP led to its widespread use in therapeutic applications. These included infections of the skin, mucous membranes, wounds, and upper and lower respiratory tract. Marchand (1893), a leading supplier of “peroxide of hydrogen” preparations, used controlled demonstrations of the superiority of his bulk preparation of peroxide (purified and stabilized) to commercial grades available at the time.2 These preparations were marketed for a variety of microbial illnesses and associated examples of clinical success.

The use of HP has had a checkered history, due to the range of concentrations (often unknown in many applications), preparations and temperatures used, as well as the interference from the presence of various soils that can react with and essentially neutralize the impact against target microorganisms. Many reports of tissue damage were due to the use of excessive concentrations of peroxide. Many failures of effectiveness were due to use of poorly stabilized preparations or inadequate temperatures for exposure or excessive amounts of contaminating soils. This was particularly true in the results of testing peroxide on bacterial spores.2

In wound application, low concentrations of unstable preparations of HP to tissues containing inactivating levels of catalase (present in blood) led to unfavorable results and general abandonment of this agent as an antiseptic; however, recent studies have begun to reevaluate the optimal use of HP in such applications as an alternative to antibiotics.5 Examination of the early literature reveals that HP was satisfactory when used as a disinfectant for inanimate materials. For example, used in low concentrations, it was considered ideal for the preservation of milk and water6 and for the sterilization of cocoa milk beverage.7 By 1950, an electrochemical process had been developed that produced pure preparations in high concentrations that were stable even at elevated temperatures and that had long shelf lives.1 Stabilization of peroxide with a small amount of acid provided a more reliable long-term concentration.

There has been an upsurge of interest in HP during the last 40 years. Yoshpe-Purer and Eylan8 used low concentrations for the sterilization of drinking water. 0.1% HP at 54°C for 30 minutes was shown to reduce the total bacterial count in raw milk by 99.999%9; and the coliform, staphylococcal, salmonellae, and clostridial counts were reduced by 100%. Investigations of 3% HP to control contamination of hospital water sources, demonstrated that a final concentration of 0.03% in water killed 1 million colony-forming units (CFUs) per milliliter of seven bacterial strains overnight, with an 80% kill in 1 hour.10 The rapid virucidal activity of HP against rhinovirus, tested both in suspension and on carriers, was demonstrated.11

In relatively high liquid concentrations (10%-25%), HP was also established as a sporicidal agent. D-values of 0.8 to 7.3 minutes at 24°C were reported with four aerobic spore strains and one anaerobic spore strain.12 Work in the Soviet Union indicated the practicability of this agent for sterilization of spacecraft; this view was supported by the studies in the United States.13 These latter studies, reported using aerobic bacterial isolates from spacecraft and achieved a complete kill of spore suspensions at the level of 100 million CFUs per milliliter by a 10% concentration at 25°C in 60 minutes. A further understanding in the difference of antimicrobial efficacy between HP is liquid and gas form was developed, demonstrating that HP was particularly effective at lower concentrations in gas form leading to the development of area fumigation and low temperature gaseous sterilization processes (see chapter 32).2 Gas and gas plasma forms became the focus on interest, particularly for single-use and reusable medical device sterilization applications (see chapter 32). Further developments focused on the optimization of HP antimicrobial activity and material compatibility by liquid formulation effects and in combination with other excipients and biocides (eg, alcohols),14,15,16 and in PAA formulations) (see chapter 6).2

Pure HP is extremely stable. Discovery of factors that cause its decomposition led to the development of effective stabilizers that deactivate contaminating materials and do not act on HP itself.1 Thus, the stability of concentrated HP can be retained when diluted to 3% if this step is carried out using clean equipment and a good grade of deionized water in clean bottles of inert material. Commercial grades of HP can contain chelants and sequestrants that minimize its decomposition. The common stabilizers include colloidal stannate and pyrophosphate, organophosphates, nitrate and phosphoric acid, and colloidal silicate.

A grade of HP (Super D®) used chiefly for the drug and cosmetic trade is highly stabilized. In the presence of added catalytic decomposing ions of aluminum, iron, copper, manganese, and chromium, a 6% solution retains 98% of its original active oxygen after being subjected to 100°C for 24 hours.17 The ordinary in-use stability of 3% HP was checked, and there was no decrease in concentration when bottles (120 mL) were opened and 10 mL was discarded each day for 12 days. Seven randomly selected shelf samples of various brands were tested according to the USP XVIII, and all were found to meet the standards for nonvolatile residues, heavy metals, and HP concentration.

The stability of a 3% HP solution that had been heated also was examined in terms of biocidal activity. The time required for an unheated 3% HP solution to eliminate a 05/mL inoculum was compared with that of a solution that had previously been subjected for 8 hours daily to 45°C for a total of 7 days. There was no significant difference in the killing times between these two preparations for seven bacterial strains and one fungus.18 Overall, commercial HP is available in a number of strengths, namely, 3%, 30%, 35%, 50%, 70%, and 90% (PeroxyChem Corp).
Special food-grade HP preparations in 35% and 50% strengths, which meet US Food and Drug Administration (FDA) safety regulations, are also available.

Peroxide is also available in dry forms, such as sodium percarbonate and sodium perborate, which release HP on contact with water. These types of formulation are used in bleaching, agricultural and environmental remediation applications (oxidizing soil and groundwater contaminants), and for generating peroxidated forms of organic acids (such as peracetic or perpropionic acid).


Mechanism of Action

The current status of the mechanism of action on the molecular level has been reviewed and summarized quite recently.2,19 The mechanism of activity is usually attributed to its reactive nature (as an oxidizing agent) and the production of highly reactive radicals by peroxide. The production of short-lived radicals can also be catalyzed in target cells or in the environment by reaction of peroxide with available transition metal ions, such iron-II, in the classic Fenton reaction:

Fe2+ + H2O2 → Fe3+ + OH + OH

It has been shown that the hydroxyl radical is a potent oxidizer of many cellular components (lipids, proteins, and nucleic acids),20 so this mechanism is commonly cited. The evidence for this Fenton-like reaction includes that bacteria grown in iron-rich media (and thus having elevated iron concentrations) were more susceptible to peroxide. This effect could be inhibited by the addition of hydroxyl scavengers such as thiourea. The use of iron chelators that can penetrate cells can protect them against killing by peroxide.21 Electron paramagnetic resonance (EPR) spin trapping of the bactericidal action of peroxides showed bactericidal activity was inhibited in the presence of antioxidants and indicated the hydroxyl radical to be part of the lethal species.22 A strong inverse correlation was found between the concentration of the trapped radical signal and the relative strength of the peroxide concentration as a bactericide. It was concluded that strong bactericides produce radicals at a much faster rate than weak ones.

The hydroxyl radical is said to be among the strongest oxidants known,1,2,3 and it is by this mechanism that HP is believed in part to elicit its antimicrobial activity. The hydroxyl radical, being highly reactive, can attack membrane lipids, DNA, and other essential cell or viral components. Transition metals, as described earlier, are believed to catalyze the formation of the hydroxyl radical; nontoxic quantity of iron, copper, chromium, cobalt, or manganese salts increased the activity of HP toward Staphylococcus aureus and Escherichia coli 100 times in one report.23 It was also suggested that, in the case of water free of metal ions, the bacteria themselves can provide the necessary metal ions.8 In the absence of metal ions in the culture medium, or if these ions are chelated with ethylene diamine tetra-acetic acid, there was no bactericidal action on E coli.24 Others suggested that the antimicrobial action of HP is particularly related to oxidation of sulfhydryl groups and double bonds in proteins, lipids, and surface membranes.25 The hydroxyl radical is also thought to contribute most of the biologic damage done by other oxidizing agents and ionizing radiation (see chapter 29).

The study of the mechanism of peroxide action has focused on the damage to DNA because this is the genetic basis of the cell and many viruses. The DNA is composed of two strands of polynucleotide phosphates. The individual nucleotide phosphates of a strand are connected by the phosphate bond to form a long strand. These strands are linked together (in a pair of strands) by hydrogen bonds between specific pairs of nucleotides, one from each strand, to form a double-stranded structure. Increased peroxide levels are associated with increased single-stranded breaks in the phosphate bonds between nucleotides in a given strand.2,19 This effect will, if unrepaired, prevent proper DNA replication or protein production. This effect has been observed for HP even at low concentrations. Cells can repair this damage, given time and nutrients. But if the damage is extensive, the result is cell death.

Observations of the exposure of bacterial cells to HP suggest two different categories of effects.2,19 At low concentrations (<3 mM), surviving cells form filaments and some loss of intracellular components is seen (suggesting some damage to cell surfaces). The rate of bacterial killing requires active cellular metabolism. Notably, DNA-repair mutants were more sensitive to this mode of killing. At much higher concentrations of peroxide (>17 mM), the cells demonstrated a much greater loss of intracellular components and a noticeable decrease in cell volume. Cells exhibit a multihit dependence of peroxide concentration and exposure time. Notably, this mode of killing does not require active metabolism, and DNA-repair mutants are not especially susceptible. It was proposed that a first mode of antimicrobial activity (low peroxide concentration) was due to DNA damage, which occurs at a low, nonlethal level during aerobic growth. A second mode (at higher concentration of peroxide) appears to be due to multicomponent damage, similar to UV-damage (see chapter 9). Others have also observed these two modes of killing.26,27 It was noted that free-radical scavengers (such as thiourea, ethanol) provided some protective effect against the higher concentration mode of killing. In addition, this could be augmented by intracellular iron content. They suggested that hydroxyl radicals are more involved high concentrations, but not lower concentrations, but this has not been confirmed. Copper-export deficient strains of E coli showed increased cellular copper inhibited antimicrobial activity and oxidative damage by peroxide.28 The higher levels of copper were associated
with both reduced activity and mutagenesis. Because copper is known to directly react with peroxide, this may be a dose-reducing phenomenon.

Direct in vitro demonstration of the damage to DNA by peroxide was shown using phage DNA incubated with ferrous sulphate and exposed to peroxide.29 Single-stranded breaks in DNA were observed, which was dose-responsive at lower peroxide concentrations (<3 mM peroxide). Excess peroxide did not produce additional nicks in DNA. The damage was reduced by ethanol, a scavenger of hydroxyl radicals. The direct DNA oxidant in the low-concentration peroxide activity was not dependent on the production of free hydroxyl radicals, but rather free ferryl radical intermediates (from a Fenton-type reaction), possibly complexed to DNA. When iron chelators are added to cells (under nonbacteriostatic conditions), a decrease in sensitivity to peroxide was observed under low peroxide (<3 mM) exposures.30 This reinforces the concept of the participation of a Fenton-like reaction under these conditions.

The DNA damage measurement in vivo was done by several groups by exposing bacteria to peroxide and measuring single-stranded breaks.31,32,33 Breaks were found, dose-dependent on the peroxide exposure, and half to most could be repaired by incubating in medium at 37°C for a period. No studies have focused on the correlation of antimicrobial activity and DNA damage, and variations in exposure conditions, media, etc, make comparisons difficult. In addition, the presence of contaminating substances (eg, protein fragments or amino acids or other soils) can have a large impact on sensitivity to peroxide. Most studies used long exposures of peroxide at low concentrations (<2.5 mM), whereas in typical usage as a disinfectant, the exposures are shorter exposures at larger concentrations (eg, 3%).19 It should also be noted that Fe2+ Fenton reaction-like damage will be less important in these applications because normally the peroxide is provided in a low iron medium. Therefore, it is anticipated that although peroxide does indeed cause damage to DNA, even at low concentrations, the killing observed in normal disinfection use must involve damage to other important macromolecules in the cell, notably proteins and membrane lipids.2 Because viruses do not have such repair mechanisms, the damage from peroxide may not be recoverable in any concentration.

Peroxide is known to react with thiol amino acids cysteine34,35,36 and methionine.37 In addition, reactions with lysine, histidine, and glycine in solution have been demonstrated. The metal-catalyzed production of hydroxyl radicals, resulting in oxidation of amino acids in proteins, has also been demonstrated,37 which can result in cleavage of the protein backbone as well as production of carbonyls. The E coli challenged with as little as 2 mM peroxide resulted in reduction in cell viability as well as an increase in protein carbonyl content.38 Yeasts are also sensitive to this treatment, resulting in excessive protein oxidation. These studies were done with low peroxide concentrations for a long exposure, so it is expected that exposure to a high concentration (eg, 3%) might cause similar damage.

Exposure of E coli to low concentrations of peroxide resulted in the expected effects as well as extensive cell filamentation.39 At higher concentrations of 17.5 mM, this filamentation did not occur, but the cell volume decreased drastically. In addition, large increases in lactate dehydrogenase activity were seen that, coupled with the decreased cell volume, suggests membrane damage and loss of cell contents was a large factor in killing the bacteria. In other studies, cell membrane permeability was observed to markedly increase in various strains of bacteria at high peroxide concentrations.40 Even in cyanobacteria exposed to peroxide, large increases in dissolved organic substances were observed as well as indicators of cell membrane damage.41 The HP has substantial effects on membrane lipids even at low concentration.


Antimicrobial Activity of Hydrogen Peroxide

The HP is active against a wide range of organisms: bacteria, yeasts, fungi, viruses, and spores (Tables 18.1, 18.2 and 18.3; Figures 18.1, 18.2and 18.3). Anaerobes are even more sensitive because they do not produce catalase to break down the peroxide. Twenty-five ppm or less was shown to prevent growth of vegetative bacteria (Table 18.4). A 3% solution of HP is rapidly bactericidal (see Table 18.3 and Figure 18.1). Even against bacteria in biofilms, it can be active at 0.5%.43 In liquid form, it is less rapid in its action against yeasts, some viruses, and especially bacterial spores (see Tables 18.1, 18.2 and 18.3); however, 6% (volume per volume [vol/vol]) HP was an effective sterilant in 6 hours.52 In general, HP has greater activity against gram-negative than gram-positive bacteria. It is less affected by pH than are many other disinfectants, such as phenols and organic acids; little difference in antimicrobial activity was shown between pH 2 and 10.53 As noted in Figures 18.2 and 18.3, an increase in concentration can have a marked effect on activity. Similar effects have been reported with gas phase HP (see chapter 32).2

Destruction of spores is greatly increased with both a rise in temperature and an increase in concentration, making HP an effective sporicide under these conditions. Under these conditions, the sporicidal activity of HP was unaffected by organic matter in the form of 25% fetal bovine serum or of salt as 3.4% sodium chloride (NaCl).54 Comparing 10% HP with seven other disinfectants against 13 species of bacteria (Table 18.5), HP was favorable in observed antimicrobial activity.55 In practical sporicidal applications with HP, high concentrations combined with high temperatures are used together to produce sterile conditions. In aseptic packaging, 35% HP at up to 80°C for 3 to 9 seconds has been used.









TABLE 18.1 Examples of antimicrobial activity of hydrogen peroxide toward bacteria, yeasts, and virusesa




































































































































































Organism


Concentration (ppm)


Lethality (min)


Temperature (°C)


Reference


Bacteria



Staphylococcus aureus


1000


60



Kunzmann42



S aureus


25.8 × 104


0.2


24


Toledo et al12



S aureus biofilm


500


1


36


Lineback et al43



Escherichia coli


1000


60



Kunzmann42



Ecoli


500


10-30


37


Nambudripad et al44



Salmonella enterica (subsp. enterica)


1000


60



Kunzmann42



Enterobacter aerogenes


500


10-30


37


Nambudripad et al44



Sarcina species


500


150


37


Nambudripad et al44



Lactococcus lactis


500


150


37


Nambudripad et al44



Streptococcus liquefaceus


500


240


37


Nambudripad et al44



Micrococcus species


30


10



Wardle and Renninger13



Staphylococcus epidermidis


30


10



Wardle and Renninger13



Pseudomonas aeruginosa biofilm


500


1


21


Lineback et al43


Yeasts



Torula species


500


180-210


37


Nambudripad and Lya45



Oidium species


500


180-210


37


Nambudripad and Lya45


Viruses



Orthinosis virus


3.0 × 101


180



Nikolov and Popova46



Rhinovirus types 1A, 1B, 7


0.75 × 101


50-60


37


Mentel and Schmidt11



Rhinovirus types 1A, 1B, 7


1.5 × 101


18-20


37


Mentel and Schmidt11



Rhinovirus types 1A, 1B, 7


3.0 × 101


6-8


37


Mentel and Schmidt11



Poliovirus type 1


1.5 × 101


75


20


Kline and Hull47



Poliovirus type 1


3.0 × 101


75


20


Kline and Hull47


a Experiments were not conducted under the same conditions and may not be comparable.



Synergism With Hydrogen Peroxide

Overall, synergism between the antimicrobial activity of biocides can be difficult to demonstrate because the activity may not be specific due to a true synergistic activity between biocides but rather each biocide working independently in the mixture as antimicrobials.56 Despite that, many reports in the literature suggest synergistic or combined antimicrobial effects with HP. The first, already suggested earlier, was reported in 1930,23 where activity against E coli and S aureus was increased 100-fold when one part of cupric and ferric ions were added to 500 parts of HP. They knew that these ions promoted the free-radical oxidation of organic compounds with HP and concluded that the bactericidal action resulted from the same cause. The supposition that free radicals were responsible for this action was reinforced when it was observed that these metals with HP in a Fenton-type reaction produced mutations in bacteria, like those produced by radiation.57

When spores of Clostridium bifermentans were treated with 100 µM of copper sulfate or 0.28 M HP at 25°C, copper alone showed a minimal effect (95% colony formation) and peroxide alone was similar (87%).58 When used together, the colony formation was significantly reduced (to 0.028%). Because HP is known to remove protein from spore coats, they tested it with dithiothreitol, which also possesses that property. Treatment with HP alone gave 93% colony formation and with dithiothreitol alone, 40%. Together, they gave 0.082%, a 500-fold reduction. It was proposed that dithiothreitol removes the protein in the spore coat that protects the spore from HP and that copper increases the rate of breakdown of HP and the rate of cleavage of peptide bonds by HP.

Studies with plasmid DNA showed that 10-2 M Cu+2 or 10-2 M HP alone did not significantly break the DNA, but a
mixture of 10-6 Cu+2 with 10-5 M HP resulted in strand breaks and inactivated transforming ability.59 The Cu+2 plus HP caused greater damage to the bases in DNA than Fe+3 plus HP.60 Working with five representative viruses, it was found that the cupric and ferric ions alone were virucidal, but this action was greatly increased with HP, particularly for copper.61 This effect extended for all of the viruses, and the investigators indicated that 0.05% Cu+2 plus 5% HP would have the virucidal activity equal to 2% glutaraldehyde. They stated that the Cu+2-HP system was more efficient in their tests than glutaraldehyde and would inactivate most or all viruses that contaminate medical devices.








TABLE 18.2 Sporicidal activity of hydrogen peroxide toward spore-forming bacteria and bacterial sporesa





















































































































































Organism


Concentration (ppm)


Lethality (min)


Temperature (°C)


pH


Commentb


Reference


Bacillus subtilis


500


420-1080


37



Mixed


Nambudripad et al44


Bacillus cereus


500


420-1080


37



Mixed


Nambudripad et al44


Bacillus megaterium


500


420-1080


37



Mixed


Nambudripad et al44


B subtilis ATCC 15411


3.0 × 101


1440


37


4.3


Spores on surface


Baldry48


B subtilis SA 22


25.8 × 104


7.3


24


3.8


Spores


Toledo et al12


Bacillus coagulans


25.8 × 104


1.8


24


3.8


Spores


Toledo et al12


Bacillus stearothermophilus


25.8 × 104


1.5


24


3.8


Spores


Toledo et al12


Clostridium sporogenes


25.8 × 104


0.8


24


3.8


Spores


Toledo et al12


B subtilis var globigii


25.8 × 104


2.0


24


3.8


Spores


Toledo et al12


B subtilis var globigii


35 × 104


1.5


24


3.8


Spores


Toledo et al12


B subtilis var globigii


41 × 104


0.75


24


3.8


Spores


Toledo et al12


B subtilis SA 22


17.7 × 104


9.4


20



Spores


Leaper49


B subtilis SA 22


17.7 × 104


0.53


45



Spores


Leaper49


B subtilis SA 22


29.5 × 104


3.6


20



Spores


Leaper49


B subtilis SA 22


29.5 × 104


0.35


45



Spores


Leaper49


B subtilis SA 22


35.4 × 104


2.3


20



Spores


Leaper49


B subtilis SA 22


35.4 × 104


0.19


45



Spores


Leaper49


a Experiments were not conducted under the same conditions and may not be comparable.

b Testing varied between studies. Mixed indicate a bacterial culture that may have contained vegetative and spore forms, spores indicate the use of a spore suspension, and all tests were done in suspension studies unless indicated on surfaces.


With the copper-HP system, the ID50 for E coli was 0.45 mg/L copper to 45 mg/L HP (1-100), whereas for glutaraldehyde, the ID50 was 4.6 mg/L.62 For Bacillus subtilis, 2% glutaraldehyde gave a 1000-fold reduction in 35 minutes, equal to that of 0.2% copper plus 5% HP. A mix of 0.2% copper plus 10% HP gave a 1000-fold decrease in 15 minutes. Against the enveloped Junin virus (Argentinian mammarenavirus), the copper-HP mixture was reported to be 50 times more virucidal than glutaraldehyde. With 10 mg/L Cu+2 to 1000 mg/L HP in media containing 5% serum, the virus was inactivated about five times faster than with glutaraldehyde.

The addition of ascorbic acid to the copper-HP system caused a large increase in the DNA base damage, much greater than with the iron-HP ascorbic acid system.60 They proposed that the copper ions bound to the DNA react with HP and ascorbic acid to generate hydroxyl radicals, which then immediately attack the DNA in a site-specific manner. With herpes simplex virus, the virucidal activity of copper was enhanced by reducing agents (HP can act as an oxidizing or reducing agent) in the following order: ascorbic acid >> HP > cysteine.63 Treatment of virus-infected cells with combinations of copper and ascorbate completely inhibited virus plaque formation to below 0.006% of the infectious virus input; it maintained 30% viability for the host mammalian cells. Other studies demonstrated the effectiveness of cupric ascorbate compared with other disinfectants, showing it to be equal or better in lower concentrations.53,55,63

The addition of other chemicals with biocidal properties to an HP solution has been shown to enhance its activity, particularly against aromatic hydrocarbons. As noted earlier, the activity can be enhanced with the addition of transition metals (although the lifetime of the reagent is short) to accelerate the production of hydroxyl radicals. Recent introduction of solutions of HP coupled with
alcohol, surfactants, and/or acids have shown enhanced antimicrobial activity and cleaning potential, particularly on surfaces.64,65 The HP is also considered to play an active role in synergism with PAA both in liquid and gas form, particularly from mode of action studies.66








TABLE 18.3 D-values with 3% hydrogen peroxide (H2O2) solution from lens disinfection studies















































































Microorganism


Min


D-valuea


Standard Error


Neisseria gonorrhoeae


b



Haemophilus influenzae


0.29


0.07


Pseudomonas aeruginosa


0.40


0.05


Bacillus subtilis


0.50


0.15


Escherichia coli


0.57


0.07


Proteus vulgaris


0.58


0.24


Bacillus cereus


1.04


0.12


Proteus mirabilis


1.12


0.33


Streptococcus pyogenes


1.50


0.25


Staphylococcus epidermidis


1.82


0.14


Staphylococcus aureus


2.35


0.18


Herpes simplex


2.42


0.71


Serratia marcescens


3.86


0.53


Candida albicans


3.99


0.54


Fusarium solani


4.92


0.54


Aspergillus niger


8.55


1.32


Candida parapsilosis


18.30


3.44


a Contamination level: 700 000 organisms per lens, 7 mL of H2O2 solution.

b Too rapid to measure.


It already has been noted that heat sharply increases the activity of HP. One explanation is that HP makes spores more sensitive to heat, and so heat may be the actual true cause of death. The HP also acts synergistically with ultraviolet (UV) radiation 0.3% HP plus UV gave 2000 times greater increase of spore kill than radiation alone and 4000 times greater than HP alone.67,68,69 Less than 1% HP in the presence of UV produced a synergistic kill, but the effect diminished, as the concentration increased. The absorption of UV by HP was postulated, as the cause for the loss of synergism. When high-intensity UV light (for 20 s) plus 2.5% HP was combined with heat up to 80°C for 60 seconds, a 5 log10 inactivation of B subtilis was obtained. These researchers attributed the antimicrobial activity to the formation of hydroxyl radicals from HP within the spores, but they explained that at higher concentrations, the decrease in activity is due to reaction of breakdown products with HP molecules outside of the
spore. The major drawback of UV with peroxide, as with UV alone, is that UV is not particularly penetrating and is limited to surface action or to clear solutions that do not absorb UV (see chapter 9). The process for sterilization by a combination of UV and HP used sequentially was patented for the commercial sterilization of packaging before filling with ultrahigh-temperature processed foods.70 A later development used a synchrotron radiation source to produce a narrow band of radiation, showing the greatest kill of B subtilis spores in the presence of HP with radiation close to 270 nm in wavelength.71






FIGURE 18.1 Examples of kinetic inactivation curves for disinfection with 3% hydrogen peroxide (in water) over time.






FIGURE 18.2 Minimal bactericidal concentration of hydrogen peroxide against time for water strains. Based on Alasri et al.50






FIGURE 18.3 Minimal sporicidal concentration of hydrogen peroxide (HP) against time for Bacillus spores. Based on Alasri et al.51

Another physical agent that has shown synergism with HP is ultrasonic energy. Experiments using ultrasonic waves in conjunction with HP found that sonication of Candida albicans and Bacillus cereus spores with 6% HP was lethal in 10 minutes, whereas the agents separately did not kill the organisms in 30 minutes’ exposure at 35°C.72 Ultrasonic energy was thought to disperse and agitate the cell aggregates, increasing surface contact with the disinfectants, increasing the permeability of the cell membrane to the disinfectant, and accelerating the rate between the disinfectant and the cell components.








TABLE 18.4 Effect of pH on the bacteriostatic activity of hydrogen peroxide (minimum inhibitory concentration in ppm)a





























pH


Pseudomonas aeruginosa ATCC 15442


Klebsiella pneumoniae ATCC 4352


Streptococcus faecalis ATCC 10541


Staphylococcus aureus ATCC 6538


5.0


5


25


25


No growth


6.5


10


25


25


5


8.0


50


25


25


5


a Based on Baldry.48



Applications of Hydrogen Peroxide

Formerly used just as an antiseptic, HP now has many other applications for preservation, cleaning, disinfection, and sterilization. In swimming pools, for example, it is used in conjunction with other disinfectants such as Baquacil®, a polymeric biguanidine; it helps to prevent bacterial growth, control algae, and clean filters without producing eye irritation, harmful products, or unpleasant odors. It is used in ultrasonic disinfectant cleaning baths for dental and medical instruments. The HP is used for odor control in sewage and sewage sludges73 and to treat landfill leachates to control microbial growth in waters that receive these discharges.74,75 In the horticultural industry, it keeps capillary feed systems for nutrient supply to plants from being plugged by algae growths.76

As a sterilant, 6% HP over 6 hours in comparison to glutaraldehyde products, which took 8 and 10 hours in different tests52; 10% HP took 1 hour and 3% HP took 2.5 hours13. Six percent HP was also an effective high-level disinfectant for flexible endoscopes to be more effective than glutaraldehyde for killing or removing B subtilis in a 10-minute contact period.77 In comparisons of 7.5% HP with 2% glutaraldehyde for disinfection of flexible endoscopes, HP needed half the exposure time of glutaraldehyde, 10 versus 20 minutes, and HP was highlighted as being less toxic to humans and the environment.78 Formulations of HP for high-level disinfection have become widely used as alternatives to aldehydes (glutaraldehyde and ortho-phthalaldehyde (OPA); see chapter 23) for these reasons.2,79

At least two interesting papers reported on the production of HP in situ. Electroplated metal coatings produced HP when in contact with electrolytes.80 It was proposed

that in the metal oxidation, the reactive O2* species is formed, which, with electrons from the metal, gives rise to the superoxide ion radical and this then to HP:








TABLE 18.5 Bacterial inactivation with hydrogen peroxide and peracetic acid compared to other biocidal agentsa,b



































































































































Bacterium


Glutaraldehyde 2%


Formaldehyde 8%


Phenol 5%


Cupric Ascorbate 0.1%


Sodium Hypochlorite 0.05%


Hydrogen Peroxide 10%


Peracetic Acid 0.03%


Bacillus cereus


>5.0 (2)


>5.0 (2)


>5.0 (2)


>5.0 (2)


>5.0 (2)


>5.0 (2)


>5.0 (2)


Clostridium perfringens


>6.3 (2)


>6.3 (2)


>6.3 (2)


>6.3 (2)


0.14 ± 0.05 (2)


>6.3 (2)


4.1 ± 0.1 (2)


Escherichia coli


>6.9 (2)


>6.9 (2)


>6.9 (2)


6.3 ± 0.8 (2)


6.2 ± 0.9 (2)


>6.9 (2)


>6,9 (2)


Listeria monocytogenes


>6.1 (2)


>6.1 (1)


>6.1 (2)


>6.1 (1)


>6.1 (2)


>6.1 (2)


>6.1 (1)


Pseudomonas aeruginosa


3.8 ± 0.2 (2)


>6.1 (3)


5.8 ± 0.6 (3)


5.6 ± 0.9 (3)


1.3 ± 0.1 (2)


>6.1 (3)


5.0 ± 1.6 (3)


Salmonella ser Typhimurium


>6.4 (3)


>6.2 (3)


>6.4 (3)


>6.4 (3)


4.1 ± 1.3 (2)


>6.4 (3)


>6.4 (3)


Shigella sonnei


>6.3 (2)


>6.3 (2)


>6.3 (2)


>6.1 (1)


>6.3 (2)


>6.3 (2)


>6.3 (2)


Staphylococcus aureus


>6.5 (3)


>6.5 (3)


>6.5 (3)


5.5 ± 1.2 (3)


4.8 ± 1.8 (2)


5.6 ± 0.7 (3)


6.6 ± 0.3 (3)


Staphylococcus epidermidis


>6.3 (3)


5.9 ± 1.1 (3)


>6.3 (3)


5.1 ± 0.1 (2)


6.3 ± 0.4 (3)


>6.3 (3)


>6.3 (3)


Vibrio cholerae


>6.4 (2)


>6.4 (2)


>6.4 (2)


>6.4 (2)


>6.4 (2)


>6.4 (2)


>6.4 (2)


Vibrio parahaemolyticus


>6.2 (1)


>6.2 (2)


>6.2 (2)


>6.2 (2)


>6.2 (2)


>6.2 (2)


>6.2 (2)


Vibrio vulnificus


>6.3 (2)


>6.3 (2)


>6.3 (2)


>6.3 (2)


>6.3 (2)


>6.3 (2)


>6.3 (2)


Yersinia enterocolitica


>6.8 (2)


>6.8 (2)


>6.8 (2)


>6.8 (2)


>6.8 (2)


>6.8 (2)


>6.8 (2)


a Calculated as -log(Td/Tw), where Td is the titer of bacteria surviving 30 min exposure at 20°C to a given disinfectant and Tw is the titer of bacteria exposed under the same conditions to water. Results are expressed either as the limit of detection when no surviving colonies were obtained or as x ± s (n) where n is the number of replicate experiments.


b Based on Sagripanti et al.55



Electroplated metal coatings of cobalt; copper; and cobalt-containing alloys of nickel, zinc, and chromium inhibited the growth of pathogenic bacteria. This inhibition was shown not to be due to the metal ions themselves, and stainless steel without the coatings was found not to be inhibitory. It was suggested that these coatings could be used to reduce bacterial contamination in hospital facilities. Low-amperage electric current (DC) electric current was also used to produce HP on the surface of electrodes used as central venous catheters.81 A zone of inhibition was demonstrated by inserting an anode and cathode into an agar plate inoculated with a lawn of bacteria. There was no zone under anaerobic conditions. The authors claimed that the electrode repels organisms from the electroconducting catheter, prevents intraluminal microbial migration, and is bactericidal to microorganisms attached to catheters, thus preventing the formation of biofilms on the electrodes.

The HP has been widely used in various forms for area disinfection (or fumigation). These include various misting systems and gas systems82,83 that claim to use HP in a gas form (known as VHP®) or in a condensed gas form.2 In these systems, liquid HP (with may include other chemicals in formulation such as silver, although the overall impact of these on antimicrobial activity is not clear) is either directly misted into the air/environment or generated into gas by heating. A typical process known as flash vaporization generates a gas by dropping a HP solution (at various concentrations between 3% and 55% in water) onto a hot surface. This forms a gas (or vapor) consisting of HP and water at the same proportion to the starting liquid). Both water and HP as vapors can readily condense into liquids (or if in a mist form go into a vapor/gas) under these conditions but dependent on the temperature in the area or on surfaces.84 Under both conditions (gas or condensed gas), HP is an effective biocide in area fumigation or sterilization applications. Under gas conditions, HP has been shown to be an effective antimicrobial with greater efficacy at lower concentrations than liquid peroxide. But under condensed conditions, peroxide will condense preferably than water, depending on the surface temperature, to lead to a high concentration of HP initially; however, it is also logical that these systems can vary in material compatibility and safety due to the presence of condensed, concentrated peroxide on surfaces.2,84 Such system applications have also been the basis for medical device or equipment disinfection85 and sterilization (see chapter 32). The HP has also been widely used in plasma sterilization systems (see chapter 32).86,87 A plasma is essentially an ionized gas, generated by providing energy to any gas such as oxygen, nitrogen, or HP (see chapter 34). Initially, sterilization systems were described that used plasmas generated from HP gas within an exposure chamber, but such systems have not been commercially successful to date.86 Despite this, many systems have been described that plasma as part of the sterilization process (see chapter 32).2 These can include using plasma energy sources to generate peroxide gas from a liquid source (essentially as an alternative to heat) and the generation of a plasma following HP gas exposure to breakdown and remove peroxide residuals from the sterilization chamber and load. The impact of plasma in these cases to antimicrobial efficacy is not considered significant, as demonstrated in studies that compared efficacy of such systems in the presence or absence of the plasma phases.88 But the overall impact to the safety of the sterilization process (in efficiency of peroxide residual removal) should not be underestimated. The use of HP in sterilization processes is further discussed on chapter 32.

The HP is widely used in food and food-contact surface disinfection. Advantages include no toxic residues, easily available and completely water soluble, and it readily degrades following application. It is used in milk and starch production, packaging, dried foods (such as eggs), and vinegars among others. Many such applications require regulatory approval, depending on the country or region. The HP is used in oral treatments (eg, in whitening and toothpaste). Although largely for the bleaching effect, it also provides antimicrobial capability. Dry sources of peroxide are also used. Examples include benzoyl peroxide, sodium percarbonate, perborate, and calcium peroxide.89 A benefit of HP is its ability to be used on various types of living tissues, including the skin and mucous membranes. Clearly at higher concentrations, peroxide can be damaging to such tissues and has health hazards, but at lower concentrations (typically less than 3% in water), it can be used to reduce microbial levels but with minimal impact on the underlying tissue. The HP is used for the disinfection/sterilization of excised tissues and allografts, such as the Allowash® process using 3% HP in combination with surfactants, alcohol, and antibiotics in a defined antimicrobial process.90,91 Certain applications have been described for use directly in the eye (at less than 1%), but overall, HP is an eye irritant and at higher concentrations can lead to irreversible eye tissue damage. The overall safety profile of HP has also seen it widely used as a preservative or cleaning/disinfection solutions/gels for contact lens and other devices that may contact the eye.92,93

But in such applications, the correct rinsing or neutralization of peroxide residuals is best practice to reduce eye irritation or damage.

The HP, as an effective oxidizing agent can be used to neutralize chemical contaminants. For example, it is used for treating pollutants (hydrocarbons, herbicide residuals,
pesticide residuals) and as a deodorizer. It has also been used as an air scrubber, to control volatile organic compound (VOCs),94 and for the neutralization of cytotoxic drug contamination in pharmacy dispensing cabinets.95 The HP can also be used for treating wastewater systems,96 typically using iron- or copper-based catalysts. It is useful form of wastewater disinfection and deodorization because bacteria are a common source of the problem.2 It is also a major bleaching agent for wood pulp, paper, and paper products, where it is used more for its whitening capability than disinfectant properties but can be used for wood or other building material disinfection.97

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Peroxygen Compounds
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