Aldehydes
Stephen A. Kelly
Sean P. Gorman
Brendan F. Gilmore
Aldehydes (from the Latin alcohol dehydrogenatum, alcohol deprived of hydrogen) are compounds containing the functional group—CHO formed as a result of oxidation of primary alcohols, where the carbonyl group is bonded to at least one hydrogen atom. The general formula for an aldehyde and the structures of the aldehyde microbicides in widespread use are shown in Figure 23.1. Currently, only three aldehyde compounds are of widespread practical use as disinfectant biocides, namely glutaraldehyde, formaldehyde, and ortho-phthalaldehyde (OPA) despite the demonstration that many other aldehydes possess good antimicrobial activity. Glutaraldehyde is one of the most commonly used biocides worldwide across a diverse range of applications, and as such is considered the archetypal compound in this biocide class and is the primary focus of this chapter. Other aldehyde biocides such as acrolein (2-propenal, acrylaldehyde) whose applications are primarily as a biocide in agriculture and industrial water supply systems (and considered a high priority air and water toxicant by the US Environmental Protection Agency [EPA]) are not considered further in this chapter.
Glutaraldehyde (pentanedial; see Figure 23.1) possesses high microbicidal activity against both bacteria and bacterial spores, fungi and their spores, and viruses. Additionally, glutaraldehyde is an effective mycobactericidal agent. Glutaraldehyde is a highly reactive saturated five-carbon dialdehyde, which interacts strongly with the bacterial cell wall, although mode of action is most likely due to interaction with proteins and enzymes (which increases with increased pH). The rate of antimicrobial activity of glutaraldehyde is pH dependent, with glutaraldehyde usually commercially obtained as an acidic solution (2%, 25%, or 50%) with 2% solutions used for disinfectant applications. Before use, the glutaraldehyde solution is “activated” (made alkaline) before use, although stable formulations that do not require activation are also available. Glutaraldehyde has a broad spectrum of activity, with rapid microbicidal action and is noncorrosive to rubber, metals, and lenses; however, its toxicity is a matter for some concern, with its use restricted or banned in some countries.
Formaldehyde (methanal; see Figure 23.1), as well as formaldehyde release agents, can be used as a preservative, as a disinfectant either in liquid or vapor form, although use is primarily as a sterilant or fumigant in the vapor phase. The UK Health and Safety Executive has indicated that inhalation of formaldehyde vapor should be presumed to be a carcinogenic risk to humans. Formaldehyde has a broad spectrum of antimicrobial activity, exhibiting lethality to bacteria and bacterial spores (although the sporicidal activity of glutaraldehyde is more rapid), fungi, and many viruses. Formaldehyde rapidly combines with proteins, and as a result, its activity may be significantly and rapidly diminished in the presence of organic matter.
The OPA (benzene-1,2-dicarboxaldehyde; see Figure 23.1), an aromatic dialdehyde biocide, is the most recently introduced aldehyde biocide in widespread use, having received clearance by the US Food and Drug Administration (FDA) in October 1999. The OPA is a high-level disinfectant with excellent, broad-spectrum antimicrobial activity including superior mycobacterial activity compared with glutaraldehyde. Due to concerns over glutaraldehyde toxicity and resistance development, OPA (0.55%) has been proposed as a glutaraldehyde replacement in high-level disinfection (a disinfection process, which kills all microorganisms except bacterial spores; see chapter 47). The OPA has numerous advantages over glutaraldehyde including low volatility and high stability over a wide pH range (pH 3-9), being virtually odorless, has good material compatibility, and is nonirritant to eyes and nasal passages. Furthermore, OPA does not require activation prior to use and glutaraldehyde-resistant bacterial strains appear not to have acquired cross-resistance. The OPA, from the studies reported to date, has excellent activity against vegetative bacteria, superior to that of glutaraldehyde, but lower activity against bacterial spores.
 FIGURE 23.1 Chemical structures of aldehyde biocides: A, Aldehyde general structure. B, Glutaraldehyde. C, Formaldehyde. D, Ortho-phthalaldehyde. |
GLUTARALDEHYDEGlutaraldehyde is a powerful biocidal agent with established reputation for the chemosterilization of equipment that cannot be sterilized by traditional physical or gaseous chemical methods (see Part IV). The first indications of its antimicrobial potential came from a survey of saturated dialdehydes in a search for an efficient substitute for formaldehyde.1 In the following year, Stonehill et al2 advocated that a suitably alkalinated solution of glutaraldehyde was rapidly sporicidal, and toward the end of 1963, a glutaraldehyde formulation was marketed for use as a chemosterilizer. The continuing interest in the compound is reflected in the numerous publications, even in recent years, on such basic aspects as its activity and mechanism of action. Emphasis is currently being applied to the toxicology of glutaraldehyde exposure, whereas much attention continues to be focused on increasing the range of applications and improvements in the activity of the compound.
Chemical Properties
Structure
Glutaraldehyde (1,5-pentanedial) appears in the official monographs of the United States Pharmacopeia and British Pharmacopoeia as glutaral concentrate and strong glutaraldehyde solution, respectively. Other synonyms used are glutaric dialdehyde and glutardialdehyde. It is usually supplied as an amber-colored liquid at an acidic pH. The saturated 5-carbon dialdehyde was first synthetized in 1908.3 As with other aldehydes, the two aldehyde groups react readily under suitable conditions, particularly with proteins.4,5 A single absorption maximum at 280 nm is exhibited by pure glutaraldehyde, although a second maximum at 235 nm is normally observed in commercial solutions because of the presence of impurities of polymers.6,7 The impurity may be removed by several methods.6,8,9,10,11 Appearance of the 235-nm absorbing polymer in pure solutions depends on storage temperature,12,13 and the rate of the polymerization depends on temperature and pH.14,15
The ratio of monomer to polymer and type of polymer present have been the subjects of numerous publications.16 Essentially, it is considered that the presence of free aldehyde groups is a prerequisite for good biocidal activity. Schemes depicting glutaraldehyde polymerization in both acid and alkaline aqueous solution are outlined in Figures 23.2 and 23.3.17 At acid pH, glutaraldehyde is in equilibrium with its cyclic hemiacetal and polymers of the cyclic hemiacetal, as proposed by Hardy et al.7,18 Increase in temperature produces more free aldehyde in acid solution, whereas in alkaline solution loss of reactive aldehyde groups is possible. When pH is raised to the neutral or basic range, the dialdehyde undergoes an aldol condensation with itself followed by dehydration
to generate α,β-unsaturated aldehyde polymers. Progression to the higher polymeric form can also occur with increased time and pH. As the pH is raised, n increases (see Figure 23.3) in value until the polymer precipitates from solution. Loss of reactive aldehyde groups could therefore be responsible for the rapid loss of biocidal activity of alkaline solutions on storage. A study into the structure of the solid aldol condensation product showed that polymerization of glutaraldehyde under basic conditions results in the formation of water-soluble and water-insoluble polymers and that these polymers contain unconjugated aldehyde, conjugated aldehyde, hydroxyl, and carboxyl groups.19 A number of other studies investigated the several products formed in the aldol condensation of glutaraldehyde and concluded that these new oligomers were glutaraldehyde trimer, pentamer, and heptamer having a 1,3,5-trioxane skeleton.20,21,22,23,24 The glutaraldehyde trimer, 2,4,6-tris(4-oxobutyl)-1,3,5-trioxane, was named “paraglutaraldehyde,” and this was proposed as being responsible for the precipitation observed occasionally during chemosterilization with alkaline glutaraldehyde.
to generate α,β-unsaturated aldehyde polymers. Progression to the higher polymeric form can also occur with increased time and pH. As the pH is raised, n increases (see Figure 23.3) in value until the polymer precipitates from solution. Loss of reactive aldehyde groups could therefore be responsible for the rapid loss of biocidal activity of alkaline solutions on storage. A study into the structure of the solid aldol condensation product showed that polymerization of glutaraldehyde under basic conditions results in the formation of water-soluble and water-insoluble polymers and that these polymers contain unconjugated aldehyde, conjugated aldehyde, hydroxyl, and carboxyl groups.19 A number of other studies investigated the several products formed in the aldol condensation of glutaraldehyde and concluded that these new oligomers were glutaraldehyde trimer, pentamer, and heptamer having a 1,3,5-trioxane skeleton.20,21,22,23,24 The glutaraldehyde trimer, 2,4,6-tris(4-oxobutyl)-1,3,5-trioxane, was named “paraglutaraldehyde,” and this was proposed as being responsible for the precipitation observed occasionally during chemosterilization with alkaline glutaraldehyde.
 FIGURE 23.2 Glutaraldehyde polymerization in aqueous acid media. A, Monomer. B, Cyclic hemiacetal. C, Acetal-like polymer. D, Monohydrate. E, Dihydrate. |
 FIGURE 23.3 Glutaraldehyde polymerization in aqueous alkaline media. Aldol-type polymer (A) progresses to a higher polymeric form (B) with time and increase pH. |
 FIGURE 23.4 Influence of time, temperature, and pH on the biocidal activity of acid and alkaline glutaraldehyde solutions: 1a,b,c and 2a,b relate to state of the molecule as described in Figures 23.2 and 23.3. |
Increased activity in acid solution through application of heat or ultrasonics25 can be explained by displacement of equilibrium toward the monomer (see Figure 23.2). A scheme relating these various factors to biocidal activity is described in Figure 23.4.
Analysis
Various methods have been used to determine glutaraldehyde concentration. Measurement of the complex absorbances resulting from glutaraldehyde reaction with 2,4-dinitrophenylhydrazine26 or 3-methyl-2-benzothiazolone hydrazone27 were early reported methods. A relationship between concentration and osmolality of glutaraldehyde solutions has also been employed.9,28 A number of methods involving measurement of the 235-nm and 280-nm absorption maxima have also been reported. The purification index (PI), defined as A235/A280, where A280 is the ultraviolet (UV) absorbance of monomeric glutaraldehyde at its λmax 280 nm, and A235 is that of polymeric glutaraldehyde at its λmax 235 nm, has been widely employed to grade glutaraldehyde products. Lower PI values represent higher purity. But care must be taken in the application of this index.21,22 A commercial glutaraldehyde product was discovered to contain an impurity that exhibits absorption at 280 nm but not at 235 nm, indicating the unreliability of the PI value. Others used a 235-nm absorbance to concentration relationship.29
A 280-nm absorbance maximum was also reported,6,13 although it was suggested that for routine laboratory use, a chemical titration method would be most suitable.6,30 Titration methods have also been studied in the reaction of
glutaraldehyde with hydroxylamine, hydrazine, and methylamine and found that a characteristic change in absorbance occurred.31 From this, a spectrophotometric method was developed for the determination of low concentrations of the aldehyde in the presence of substrates absorbing in the same wavelength region. A microplate photometric method based on N-methylbenzo-thiazol-(2)-hydrazone and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole has been developed to measure various aldehydes, including glutaraldehyde, in disinfectant solutions.32 Other methods of analysis have been developed for specific reasons such as the determination of glutaraldehyde at low levels in hospital air.33 Chromatographic methods have also been described. These gas chromatographic34,35 and high-performance liquid chromatographic (HPLC) methods22 have compared favorably with UV methods. It is advised that the chromatographic methods should be used in preference to the UV-absorption (235/280 nm) or UV spectrophotometric method (hydroxylamine).23,36 A HPLC method for glutaraldehyde determination was defined and validated by derivatization using 2,4-dinitrophenylhydrazine. The method was validated for concentration ranging from 1.25 mg/L with a detection limit of 0.2 mg/L.37
glutaraldehyde with hydroxylamine, hydrazine, and methylamine and found that a characteristic change in absorbance occurred.31 From this, a spectrophotometric method was developed for the determination of low concentrations of the aldehyde in the presence of substrates absorbing in the same wavelength region. A microplate photometric method based on N-methylbenzo-thiazol-(2)-hydrazone and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole has been developed to measure various aldehydes, including glutaraldehyde, in disinfectant solutions.32 Other methods of analysis have been developed for specific reasons such as the determination of glutaraldehyde at low levels in hospital air.33 Chromatographic methods have also been described. These gas chromatographic34,35 and high-performance liquid chromatographic (HPLC) methods22 have compared favorably with UV methods. It is advised that the chromatographic methods should be used in preference to the UV-absorption (235/280 nm) or UV spectrophotometric method (hydroxylamine).23,36 A HPLC method for glutaraldehyde determination was defined and validated by derivatization using 2,4-dinitrophenylhydrazine. The method was validated for concentration ranging from 1.25 mg/L with a detection limit of 0.2 mg/L.37
Reaction With Proteins
It is likely that several reactions occur between glutaraldehyde and proteins, giving rise to several products. Several studies of glutaraldehyde interaction with proteins in vitro have been published,38,39,40 shedding some light on this subject. The composition/purity of the glutaraldehyde solution is known to dictate the type of reaction products formed with amino acids.41 The rate of reaction with protein is pH dependent and increases considerably over the pH 4 to 9 range,5 and it is known that the reaction gives rise to a product (or products) that is stable to acid hydrolysis and a chromophore with an absorption maximum at 265 nm. The stability of the cross-linkages to acid hydrolysis rules out Schiff base formation.42 Aldol-type polymers formed in alkaline solution react with amino groups to give an imino bond stabilized by resonance with the ethylenic double bond (Figure 23.5), and it was proposed that glutaraldehyde does not react with proteins in its free form but as an unsaturated polymer.43 On the other hand, the tanning actions of aqueous solutions of purified and unpurified glutaraldehyde are almost identical,18,44,45,46 indicating that a possible cross-linking reaction does not depend on the initial presence of unsaturated compounds.
In a study of the mechanism of cross-linking of proteins employing bovine pericardium, it is suggested that glutaraldehyde fixes primarily the surface of the fibers and creates a polymeric network that hinders the further cross-linking of the interstitium of the fiber.47
 FIGURE 23.5 Reaction of aldol-type polymers of glutaraldehyde with amino acids. |
Proteins are composed of amino acids, some of which contain free amino groups that readily react with glutaraldehyde. In particular, lysine possesses an ε-amino group that is the principal side chain of the molecule and therefore accessible to glutaraldehyde.48 A further mechanism was proposed to explain the nature of glutaraldehyde-protein cross-links.46 The reaction of free aldehyde with a primary amine of the protein is followed by condensation of additional free glutaraldehyde and leads to the formation of a 1, 3, 4, 5 substituted pyridinium salt analogous to the amino acid desmosine (Figure 23.6). This mechanism ties in with the observation of a new absorption peak at 265 nm as proteins are cross-linked, and the lability of pyridinium type cross-links to alkaline hydrolysis is similar to protein-glutaraldehyde cross-links treated in the same manner.49 The pyridinium linkage is not the only type of cross-link in glutaraldehyde-treated proteins, but it most likely represents a significant portion of these, with other possibilities existing. The reaction of glutaraldehyde with adenosine, cytidine, and guanosine and with the equivalent deoxyribonucleosides has been investigated.50 Multiple products were seen in HPLC for adenine and guanine nucleosides. In each nucleoside, the site of reaction was shown to be the exocyclic amino group. Chemically, the bonds were thought to be Schiff bases.
Antimicrobial Properties
Glutaraldehyde displays a broad spectrum of activity and rapid rate of kill against the majority of microorganisms (Table 23.1). Glutaraldehyde as a chemical sterilant is capable of destroying all forms of microbial life including bacterial and fungal spores, tubercle bacilli, and viruses. Similarly, it has been classified as a high-level disinfectant capable of producing sterility if the exposure was long enough.54
 FIGURE 23.6 A protein-pyridinium cross-link resulting from glutaraldehyde reaction with protein. |
TABLE 23.1 Lethal effects of aqueous 2% alkaline glutaraldehyde solutionsa | ||||||||||||||||||||||||||||||||||||
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TABLE 23.2 Comparative sporicidal activities of some aldehydes | ||||||||||||||||||||||||
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Bacterial Spores
The ability of glutaraldehyde to kill bacterial spores is, perhaps, its most important property. Useful sporicidal activity is a relatively rarer property of chemical disinfectants, which are often bactericidal only (see chapter 5). Glutaraldehyde is the only aldehyde to exhibit excellent sporicidal activity (Table 23.2), an activated 2% solution having a greater effect than 8% formaldehyde against a range of bacterial spores (Table 23.3). Increasing attention has been focused on the development of alternative disinfectants having useful sporicidal activity. But difficulties remained in balancing useful activity with inherent disadvantages such as corrosivity, inactivation by organic matter and toxicity, which are all too often present. A comparative study into the sporicidal activity of several glutaraldehyde, sodium dichloroisocyanurate, and peroxygen disinfectants typified the situation.55 Bacillus subtilis NCTC 10073 spores were rapidly eliminated (<1 h) in the absence of blood by the various chlorinecontaining compounds (ranging from 1200 ppm to 5750 ppm Cl2). But presence of blood effectively negated this activity. Peroxygen disinfectants were variable in their activity, but the peracetic acid disinfectant tested proved effective in the presence and absence of blood at the concentrations tested. A glutaraldehyde-phenate and a “long-life” glutaraldehyde disinfectant were also examined. These were tested at 30 days and 28 days, respectively, and found to much slower in action (>1 h) than the other disinfectants tested but did retain their activity in the presence of blood.
TABLE 23.3 Comparison of the sporicidal activity of formaldehyde and glutaraldehydea | ||||||||||||||||||||
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The time required for sterilant process by a chemical agent is based on the killing time achieved by the agent against a reasonable challenge of resistant spores. At the common use-dilution of 2%, a glutaraldehyde formulation was capable of killing spores of Bacillus and Clostridium in 3 hours.2,56 Others reported a 99.99% kill of spores of Bacillus anthracis and Clostridium tetani in 15 and 30 minutes, respectively.57 It was apparent from these results that not all species were equally susceptible, and of those organisms tested, Bacillus pumilis was the most resistant. Studies of the sporicidal activity of 2% glutaraldehyde against B subtilis var globigii, Geobacillus stearothermophilus, and Clostridium difficile found that the aerobic species, normally chosen for test purposes, survived for 2 hours, but C difficile was killed in under 10 minutes.58 Overall, B subtilis spores are found to be the most resistant to treatment with glutaraldehyde.59 Using the Association of Official Analytical Chemist (AOAC) sporicidal test and vacuum-dried spores, 10 hours was necessary for complete kill. In an investigation including nine glutaraldehyde formulations, it was reported that all were effective against a spore suspension of B subtilis var
globigii in 3 hours or less.60 A similar result was obtained with a challenge of 106 spores dried onto aluminum foil. A 3-hour exposure was recommended at that time to be sufficient for practical purposes, particularly as spores are infrequent on clean medical equipment. Other work, using similar time-survivor measurements and aqueous suspensions of B subtilis spores, indicated that a 3-hour period gave approximately a 6 log drop in viable count.61,62,63,64
globigii in 3 hours or less.60 A similar result was obtained with a challenge of 106 spores dried onto aluminum foil. A 3-hour exposure was recommended at that time to be sufficient for practical purposes, particularly as spores are infrequent on clean medical equipment. Other work, using similar time-survivor measurements and aqueous suspensions of B subtilis spores, indicated that a 3-hour period gave approximately a 6 log drop in viable count.61,62,63,64
Antibacterial Activity
Vegetative bacteria are readily susceptible to the action of glutaraldehyde. As shown in Table 23.4, a 0.02% aqueous alkaline solution was rapidly effective against gram-positive and gram-negative species, and a 2% solution was capable of killing any vegetative species, including Staphylococcus aureus, Proteus vulgaris, Escherichia coli, and Pseudomonas aeruginosa within 2 minutes.2 Complete kill in 10 minutes of E coli suspensions (2 × 108 cells/mL) by 100 µg/mL alkaline glutaraldehyde was reported, compared with a 45% kill produced by acid solution.69 Glutaraldehyde preparations passed the Kelsey-Sykes capacity test with and without yeast, using P aeruginosa as the test organism, compared with hypochlorite under the concentrations tested, which failed the test when yeast was added.60 Using stainless steel penicylinders, neoprene “O” rings, and polyvinyl tubing as carriers for a range of organisms including P aeruginosa and Mycobacterium smegmatis to simulate in-use conditions for the sterilization of instruments, catheter tubing, and anaesthetic equipment, a glutaraldehyde preparation was more effective on all three carriers than a cetrimide/chlorhexidine gluconate disinfectant, which was only partially effective.70
Mycobacteria
The tubercle bacillus is more resistant to chemical disinfectants than other nonsporing bacteria (see chapter 3). Because glutaraldehyde is widely used for the cold disinfection of respiratory equipment that may be contaminated with tubercle bacilli, it must have good activity against these organisms. Earlier reports of glutaraldehyde activity against mycobacteria have been conflicting, with some claiming good mycobactericidal activity.2,56,64,71 Others have shown a slow action against Mycobacterium tuberculosis,57 being less effective than formaldehyde or iodine72 or hypochlorite.73 Two percent glutaraldehyde formulations for 10 to 30 minutes were recommended for the chemical disinfection of fiberoptic endoscopes, where an iodophor formulation was less effective against the tubercle bacillus.74 The use of glutaraldehyde as an alternative to other disinfectants in tuberculosis laboratory discard jars was investigated.75 A 5 log reduction in viability of clinical isolates of M tuberculosis was obtained within 10 to 30 minutes at 25°C using alkaline glutaraldehyde, even in the presence of neutralizing materials such as swab sticks and sputum. A possible explanation for these varying conclusions was suggested, which found the official AOAC test procedure to be nonquantitative and to lack precision and accuracy.76 It was concluded that use of carriers increased variability of results and suggested a new test method for mycobacterial efficacy. But another interpretation is that the cross-linking of mycobacterial cells onto the surface in the presence of extraneous materials (contaminating protein) may protect the cells from inactivation and, although still viable, are unable to grow unless physically dislodged (see chapter 61). The importance of accurate temperature control was also highlighted.77 A significant change in rate of kill of Mycobacterium bovis bacille Calmette-Guérin by glutaraldehyde was observed as temperature was increased from 20°C to 25°C. Clumping of bacilli was proposed as another cause of erroneous results.
TABLE 23.4 Susceptibilities of nonsporing bacteria to 0.02% aqueous alkaline glutaraldehydea | |||||||||||||||||||||||||||||
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The choice of organism for use in mycobactericidal tests is also controversial. Investigators have often used M smegmatis in suspension and a variety of carriers in the presence of sputum to assess mycobactericidal activity of disinfectants.78 In these tests, glutaraldehyde produced in excess of a 6 log reduction in viable count after 1 minute of contact; however, others suggested M smegmatis is more susceptible to disinfectants than M tuberculosis, and therefore, its use as a test organism is not appropriate.79 These authors suggested using Mycobacterium terrae,
which produced results more closely related to M tuberculosis in the suspension test employed. Once again, a glutaraldehyde formulation was found to be rapidly mycobactericidal. Variation in resistance to glutaraldehyde was shown by different strains of mycobacteria, with strains of Mycobacterium avium and Mycobacterium intracellulare requiring over 20 and 40 minutes, respectively, to achieve a 99% kill.80 Differences in sensitivity to glutaraldehyde between laboratory strains and clinical isolates had been observed previously.81 The antimycobacterial activity of glutaraldehyde is summarized in Table 23.5. But it is interesting to note increasing reports of high-level resistance to glutaraldehyde in atypical mycobacteria strains (Mycobacterium chelonae, Mycobacterium massiliense) that have been isolated from environmental conditions (eg, water and equipment) and from patient infections.79,86,87,88,89 Some of these strains were also reported to be cross-resistant to other aldehydes (OPA) and demonstrated increased virulence in animal studies.89,90,91 The resistance mechanisms have been studied and is most likely to be due to the lack of appreciable or sensitive protein molecules at the surface of the mycobacterial cell wall in these strains, thereby limiting the activity or penetrability of glutaraldehyde.92
which produced results more closely related to M tuberculosis in the suspension test employed. Once again, a glutaraldehyde formulation was found to be rapidly mycobactericidal. Variation in resistance to glutaraldehyde was shown by different strains of mycobacteria, with strains of Mycobacterium avium and Mycobacterium intracellulare requiring over 20 and 40 minutes, respectively, to achieve a 99% kill.80 Differences in sensitivity to glutaraldehyde between laboratory strains and clinical isolates had been observed previously.81 The antimycobacterial activity of glutaraldehyde is summarized in Table 23.5. But it is interesting to note increasing reports of high-level resistance to glutaraldehyde in atypical mycobacteria strains (Mycobacterium chelonae, Mycobacterium massiliense) that have been isolated from environmental conditions (eg, water and equipment) and from patient infections.79,86,87,88,89 Some of these strains were also reported to be cross-resistant to other aldehydes (OPA) and demonstrated increased virulence in animal studies.89,90,91 The resistance mechanisms have been studied and is most likely to be due to the lack of appreciable or sensitive protein molecules at the surface of the mycobacterial cell wall in these strains, thereby limiting the activity or penetrability of glutaraldehyde.92
TABLE 23.5 Mycobacterial activity of glutaraldehyde | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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In conclusion, claims of glutaraldehyde activity against mycobacteria must be regarded with caution, taking into account the method and temperature used in the assessment and the criteria employed to measure success or failure of disinfection. All reports indicate activity of glutaraldehyde against these organisms; it is the rate of kill that is affected by method and conditions used. The practical implications of these reports are that adequate time for decontamination of equipment must be allowed.
Antifungal Activity
Glutaraldehyde has been shown to exhibit potent activity against a range of fungi, including the dermatophytes Trichophyton interdigitale and Microsporium gypseum, the yeasts Candida albicans and Saccharomyces cerevisiae, the common spoilage molds Mucor hiemalis, Rhizopus stolonifer, and Penicillium chrysogenum, and the resistant fruit spoilage mold Byssochlamys fulva.93,94,95,96 Fungicidal activity of a 0.5% glutaraldehyde solution is illustrated in Figure 23.7 against spores of Aspergillus niger. This fungus was found to be more resistant than other fungi to glutaraldehyde,57,96 presumably due to its greater intrinsic tolerance to microbiocides than other fungi (see chapter 3).92 But in common with a range of other fungal species, both mycelial growth and sporulation are inhibited by 0.5% alkaline glutaraldehyde, whereas spore swelling is entirely halted by a 0.5% solution. A niger and
Aspergillus fumigatus were found to be the most resistant fungi encountered in a comparative study of fungicidal activity of disinfectants.97 Sonacide® (an acid-based glutaraldehyde formulation) was effective against both fungi; however, a glutaraldehyde-phenate mixture (Sporicidin®) was not effective, even after 90 minutes of contact.
Aspergillus fumigatus were found to be the most resistant fungi encountered in a comparative study of fungicidal activity of disinfectants.97 Sonacide® (an acid-based glutaraldehyde formulation) was effective against both fungi; however, a glutaraldehyde-phenate mixture (Sporicidin®) was not effective, even after 90 minutes of contact.
 FIGURE 23.7 Effect of 0.5% acid or alkaline glutaraldehyde formulation on spores of Aspergillus niger. From Gorman and Scott.96 |
Antiviral Activity
Reliable scientific evidence of viricidal activity of disinfectants became increasingly necessary during the 1980s and 1990s because more information becomes available implicating direct contact with infected material as a significant means of transmission of infection. Two publications, while developing a test method to determine viricidal activity of disinfectants, have confirmed the antiviral activity of glutaraldehyde. When using an ultrafiltration dilution technique to separate disinfectant from virus, it was demonstrated that 2% glutaraldehyde was rapidly viricidal to poliovirus type 1.98 A surface test in which a standard challenge of 3 × 107 plaque-forming units of herpes simplex was allowed to dry onto coverslips before exposure to a range of disinfectants was also investigated99; no viable virus was recovered after 1 minute of contact with 2% alkaline glutaraldehyde, and overall glutaraldehyde and ethanol or isopropanol were the most active of all the agents tested. A number of earlier reports showed that glutaraldehyde was effective against a range of viruses,100,101,102,103 even in the presence of high levels of organic matter.104,105 Enveloped lipophilic viruses usually show significantly less resistance than the nonlipid viruses (see chapter 3). The nonlipid enteroviruses—polio, echo, and coxsackie—showed greater resistance to disinfection with glutaraldehyde than other virus groups.106 A potentiated acid glutaraldehyde formulation was also shown to have weak activity against coxsackievirus B5 and echovirus 11 in studies107,108 and to be less effective than ethanol or a chlorine-based disinfectant against reovirus 3.109
Many studies concentrated on hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The HBV infection continues to be a major health hazard, especially among health care professionals. Because of the risks to personnel and initial lack of data relating to disinfectant activity toward HBV, infection-prevention guidelines tended to recommend only strong disinfectants such as glutaraldehyde or high concentrations of hypochlorites for the treatment of HBV-contaminated material. These recommendations are supported by evidence that glutaraldehyde is capable of inactivating HBV antigen110 and destroying HBV infectivity.111,112 Glutaraldehyde was shown to produce a time and concentration-dependent reduction in hepatitis A viral titer and a decrease in antigenicity.113 Further studies with HIV continued to support the efficacy of glutaraldehyde against the virus, displaying sensitivity to chemical disinfection similar to that of other enveloped viruses.114 Reverse transcriptase was used as an indicator of viral inactivation, and although this assay was demonstrated to be less sensitive than measurement of infectious virus titer, it was also concluded that HIV behaved similarly to other enveloped viruses.115
Rotaviruses are responsible for numerous outbreaks of acute gastroenteritis in young children. The possible risk of transmission of these viruses by contaminated hand or fomite contact could be reduced by good disinfection practice. In an evaluation of disinfectant activity against human rotaviruses, 2% alkaline glutaraldehyde solution in the presence of an organic load produced at least a 3 log reduction in virus plaque titer within 1 minute in a suspension test116 and using virus-contaminated inanimate surfaces.117 Rotaviruses, similar to polioviruses, represent the nonenveloped viruses that generally have a greater resistance to microbicide inactivation. In this case, these viruses can demonstrate variable viricidal activity with glutaraldehyde due to their external protein capsid structure that may or may not react with glutaraldehyde (see chapter 3). Parvoviruses, for example, show variable activity with both glutaraldehyde and OPA but over time do show variable activity.118 Recently, investigations claiming the unusual lack of activity again human papilloma viruses119 have not been substantiated and are likely due to test method aberrations rather than lack of antiviral activity (Yarwood, personal communication, September 2019).
Resistance
Reports in the literature of resistance of microorganisms to glutaraldehyde resulting in contamination and occasionally infection may be attributed to two factors. First, where reference is made to outbreak of infection or spread of contamination through use of glutaraldehyde,120,121,122 the agent was invariably employed as a disinfectant rather than as a sterilizing agent. The short time available between patients on, for example, endoscopy lists, has
necessitated a reduction in contact time for decontamination in many instances, and this has inevitably led to the reported cases. Indeed, 45 minutes of glutaraldehyde contact was necessary to achieve sterilization of heavily contaminated flexible-fiber bronchoscopes.123 It is also likely that in many of these cases, the disinfectant was not used appropriately or indeed cross-contamination following disinfection due to the use of tap water (which is often contaminated with pathogens are low levels at the point of use).
necessitated a reduction in contact time for decontamination in many instances, and this has inevitably led to the reported cases. Indeed, 45 minutes of glutaraldehyde contact was necessary to achieve sterilization of heavily contaminated flexible-fiber bronchoscopes.123 It is also likely that in many of these cases, the disinfectant was not used appropriately or indeed cross-contamination following disinfection due to the use of tap water (which is often contaminated with pathogens are low levels at the point of use).
A second factor that must also be considered is intrinsic microorganism resistance. The TM strains of M chelonae survived 60 minutes of exposure to 2% alkaline glutaraldehyde, although no survivors of ATCC strains of M chelonae or Mycobacterium fortuitum were detected in fluids assayed at 2 minutes of contact time.81 With 0.2% glutaraldehyde, both TM and ATCC strains showed survivors at 96 hours of exposure time. M chelonae organisms have been reported as intrinsic contaminants of porcine prosthetic heart-valve tissues treated and stored, respectively, in 1% and 0.2% glutaraldehyde solutions.124
Disinfectant solutions used to treat materials and equipment for patient use must therefore be carefully evaluated in terms of their potential for harboring rather than eliminating contaminants. Glutaraldehyderesistant nontuberculous mycobacteria are becoming increasingly problematic for health care organizations. More than 2000 cases of infection from a single strain of glutaraldehyde-resistant Mycobacterium abscessus subsp massiliense (BRA100) were reported in Brazil between 2004 and 2008.87,125 In a subsequent study, clinical BRA100 isolates were recovered following postsurgical infection. These strains once again proved highly resistant, with minimum inhibitory concentration (MIC) values of 8% glutaraldehyde. These isolates were susceptible to both OPA and peracetic acid, suggesting glutaraldehyde should be replaced with OPA or other solutions for highlevel disinfection.126 Nosocomial infections have also been traced to endoscopes, bronchoscopes, and dialyzers in the Netherlands, Japan, the United Kingdom, and the United States, with glutaraldehyde-resistant M chelonae and M abscessus isolates identified as the causative organisms.88,89,127,128 As discussed earlier, changes in the protein availability on the surfaces of these strains appear to be the major mechanism of resistance to glutaraldehyde, and in some cases was also cross-resistant to OPA.88,92 Others recently reported the emergence of glutaraldehyde-resistant P aeruginosa in a health care setting. Two strains of glutaraldehyde-resistant P aeruginosa were found in the rinsing water and drain of an endoscope reprocessor. A number of patients with lower respiratory tract and bloodstream infections were identified as having possible epidemiologic links to the resistant P aeruginosa strains.129
Efflux mechanisms have been shown to contribute to glutaraldehyde resistance in both Pseudomonas fluorescens and P aeruginosa biofilms. Transcriptomic analysis revealed genes involved in multidrug efflux were induced in the presence of sublethal concentrations of glutaraldehyde. Furthermore, glutaraldehyde activity was potentiated by the addition of efflux pump inhibitors (EPIs), resulting in significant improvements in biofilm inactivation,130 but this mechanism of resistance is difficult to understand considering the mode of action of glutaraldehyde as a microbicide. The P fluorescens was also used as a model organism to investigate the genetic response elucidating tolerance to glutaraldehyde in reused wastewater (produced water). The altered resistance profile of microorganisms to glutaraldehyde was determined to be due to increased salinity of the produced water, with genes involved in osmotic stress, energy production and conversion, membrane integrity, and protein transport found to facilitate bacterial survival following biocide treatment.131
As with resistance to similar antimicrobial compounds, resistance to glutaraldehyde is considered multifactorial. Outer membrane structure and composition as well as the presence of integrons and modulators of biofilm formation all appear to play a role in resistance development.130,132,133 Although examples of glutaraldehyde resistance are increasing, accepted breakpoint values to determine resistance do not currently exist. An MIC value of >4000 mg/L (0.4%) has been suggested for Bacillus strains, which may be suitable for other species to describe resistance.134
Sporicidal results66 have shown that a spore population of B subtilis treated with alkaline glutaraldehyde, and presumed dead, can be revived in defined germination medium following removal of the outer coat layers of the spore with selective agents and by application of ultrasonic energy (Table 23.6). A small proportion may be recovered after 3 to 10 hours of contact with 2% glutaraldehyde solutions by application of ultrasonic energy, lysozyme, and protein-denaturing agents such as dithiothreitol and urea-mercaptoethanol. This revival may be considered academic in nature, but it has implications in practice, especially in view of the differences in resistance exhibited by natural and subcultured populations as suggested earlier. It is likely that viable spores have been protected from complete inactivation due to cross-linking and entrapment within contaminated surfaces. Furthermore, there is a risk attached to the use of sublethal concentrations of glutaraldehyde (ie, less than 2%) for disinfection or sterilizing purposes under soiled conditions. Such concentration levels may arise not only from in-use dilution but also from polymerization of alkaline solutions of glutaraldehyde and presence of organic matter.135 The presence of various types and amounts of organic and inorganic materials, as well as changes in pH, may lead to adsorption, alteration, or inactivation of the disinfectant, significantly reducing recommended effective concentrations. Also, substandard preparation of the “activated” disinfectant, contamination of solutions,
failure to replace solutions that have deteriorated on standing, or even dilution of residual glutaraldehyde solution may all modify the outcome of disinfection. Illustrating this point, the “reuse” of glutaraldehyde solutions has been investigated.136 Samples of the disinfectant were collected from baths over the recommended 14 day reuse period, analyzed, and assessed for activity against M bovis and hepatitis A virus. Glutaraldehyde levels dropped to approximately 1% (from 2.25% initial) by 14 days. Due attention should therefore be exercised in the use of glutaraldehyde, as with any disinfectant, to avoid such an occurrence especially in view of the resurgence of pathogens such as mycobacteria, viruses, and antibiotic-resistant bacteria.
failure to replace solutions that have deteriorated on standing, or even dilution of residual glutaraldehyde solution may all modify the outcome of disinfection. Illustrating this point, the “reuse” of glutaraldehyde solutions has been investigated.136 Samples of the disinfectant were collected from baths over the recommended 14 day reuse period, analyzed, and assessed for activity against M bovis and hepatitis A virus. Glutaraldehyde levels dropped to approximately 1% (from 2.25% initial) by 14 days. Due attention should therefore be exercised in the use of glutaraldehyde, as with any disinfectant, to avoid such an occurrence especially in view of the resurgence of pathogens such as mycobacteria, viruses, and antibiotic-resistant bacteria.
TABLE 23.6 Revival of alkaline glutaraldehyde-treated Bacillus subtilis spores after coat removal and resuscitationa | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Initial viable spore count: 2 × 107 colony-forming units/mL
Exceptional resistance to sterilization and disinfection is exhibited by the causative agent of scrapie and other prion agents (see chapter 68).137,138 Scrapie agent was shown to be more resistant than other organisms to glutaraldehyde and was not fully inactivated by 12.5% glutaraldehyde in 16 hours at 4°C.138 In a more recent study, scrapie infectivity survived exposure to 12.5% unbuffered glutaraldehyde (pH 4.5) for 16 hours.139 Furthermore, the causative agent for Creutzfeldt-Jakob disease (CJD) was not inactivated following 14-day exposure to 5% glutaraldehyde at pH 7.3.140 Results are not unexpected due to the proteinaceous nature of prions and their associated structures (see chapter 68).
Microbial biofilm formation on medical devices is an acknowledged problem in respect to antimicrobial agent resistance. Whereas glutaraldehyde 2% has been shown to eradicate laboratory-grown biofilm cells of P aeruginosa within 1 minute,141 concerns justifiably exist as to efficacy against biofilm formed on flexible endoscopes in automatic machines. Griffiths et al86 examined glutaraldehyde 2% against M chelonae isolates from automatic machines and which had been exposed to selective pressure of disinfectant usage. These isolates were extremely resistant to glutaraldehyde. It was concluded that strict attention must be placed on sessional and regular cleaning of the machines to prevent biofilm formation. The effect of glutaraldehyde disinfection on microbial biofilms from a range of species was recently investigated.142 Glutaraldehyde was not capable of preventing the survival of bacteria in biofilms of Salmonella ser Typhimurium, E coli, Streptococcus mutans, or Bacteroides fragilis. In a recent study on glutaraldehyde disinfection of S aureus and P aeruginosa biofilms from endoscopes, cells in residual biofilm remained viable after treatment with 2% glutaraldehyde.143 Environmental issues surrounding glutaraldehyde use also remains a concern. Environmental partitioning studies suggest glutaraldehyde tends to remain in the aquatic compartment in wastewater, with little tendency to bioaccumulate. This exposes aquatic microorganisms to sublethal concentrations of glutaraldehyde, which may result in the proliferation of tolerant or resistant microbes.144,145
Mechanism of Action
Interactions With Bacterial Cell Constituents
Glutaraldehyde-protein interactions, as described earlier, indicate an effect of the dialdehyde initially on the surface of bacterial cells. Conclusions from a range of mode-ofaction studies indicate a powerful binding of the aldehyde to the outer cell layers. Earlier studies found that the dialdehyde reacted with 30% to 50% of the ε-amino groups in isolated peptidoglycan, and it was proposed that two tripeptide side chains could be joined when free ε-amino groups are available.146 Treatment of E coli whole cells and isolated walls with alkaline glutaraldehyde greatly reduced, or completely prevented, lysis by 2% sodium lauryl sulphate at 35°C to 40°C,69 and pretreatment of S aureus and P aeruginosa cells with glutaraldehyde reduces subsequent lysis by lysostaphin and EDTA-lysozyme.147,148 Strengthening of the outer layers of spheroplasts and protoplasts by glutaraldehyde has also been reported.149,150 Cell agglutination, shown to occur on addition of glutaraldehyde to various microorganisms, was suggested to be due to the formation of intercellular bonds, thus confirming the hypothesis of a preferential action of glutaraldehyde on the outer layers of the cells.151 But the biocidal effect of glutaraldehyde is unlikely to be due to a sealing of the cell envelope alone.152,153 Transport of a low-molecular-weight amino acid, α-amino-isobutyric-acid-1-14C (14 C-AIB), was compared in glutaraldehyde-treated and untreated cells of E coli and found to be reduced by only 50% in treated cells.
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