ortho-Phthalaldehyde: Mechanisms of Action against Mycobacteria

Chapter 9

0rtho-Phthalaldehyde: Mechanisms of Action against Mycobacteria Charles G. Roberts and Chris R. French

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Advanced Sterilization Products, Biocides Research, A Johnson and Johnson Company, 33 Technology Drive, Irvine, C A 92618

In recent years, ortho-phthalaldehyde (OPA) solutions have emerged as alternatives to glutaraldehyde solutions for highlevel disinfection of semicritical medical devices. The increased use of OPA-based disinfection solutions is due in part to the antimicrobial activity of OPA against glutaraldehyde-resistant mycobacteria. In this chapter we review the available information on the mechanisms of action of OPA against mycobacteria, which are well known for their resistance to many different chemical germicides. The unique cell wall architecture of the mycobacteria, measurements of cell surface hydrophobicity, and resistance to aldehyde-based disinfectants are discussed.

182

© 2007 American Chemical Society

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


183 Disinfection can be defined as a chemical process that is capable of destroying most types of disease-producing microorganisms on inorganic surfaces. Disinfectants are commonly divided into different classes based on their effectiveness against different microorganisms. Three disinfectant categories are typically recognized: "low-level," which kills most vegetative bacteria and some viruses and fungi, but not mycobacteria or bacterial spores; "Intermediate-level," which kills vegetative bacteria, most viruses and all fungi, but does not kill spores; and "high-level," which kills all microorganisms except high numbers of spores. A classification system for medical instrumentation devised in the late 1960's by Earle Spaulding is commonly used to determine the appropriate methods for preparation of medical instruments prior to use. The clinical utilization of the instrument determines the level of disinfection required. The three categories of instruments or devices are: "critical," introduced into normally sterile areas of the body or into the vascular system; "semicritical," which contact intact mucous membranes and usually do not penetrate body surfaces; and "noncritical," which contact only intact skin. High-level disinfection is the minimum treatment recommended for the reprocessing of semicritical medical instruments. Glutaraldehyde-based high-level disinfectants (HLDs) were first introduced for this purpose in 1963 and have been the disinfectants most often used for high-level disinfection since that time (7). In the 1970's, reports surfaced of mycobacteria that exhibit increased resistance to these glutaraldehyde-based disinfectants. In response to the threat posed by glutaraldehyde-resistant mycobacteria, ortAo-phthalaldehyde solutions (OPA) have emerged as an alternative to glutaraldehyde solutions for high-level disinfection of reusable medical devices, especially flexible endoscopes (2). Bruckner et al. disclosed in a 1990 patent (5) a sterilizing and disinfecting solution containing a saturated dialdehyde and an aromatic dialdehyde. The aromatic dialdehyde contributed to a more rapid kill of Mycobacterium bovis (BCG) at 20° C without diminishing the capability of the solution to destroy other microorganisms, including bacterial spores. The preferred aromatic dialdehyde was found to be phthalaldehyde due to its reasonable solubility in water and tuberculocidal activity. Examples showed that when several aromatic dialdehydes, including phthalaldehyde, were added to 2% glutaraldehyde solutions, the solutions demonstrated enhanced tuberculocidal activity. A separate example showed that phthalaldehyde had excellent tuberculocidal activity by itself. Also demonstrated was that with as low as 0.005% (w/w) phthalaldehyde, solutions of 2% glutaraldehyde are tuberculocidal within 30 minutes at 20° C. Similarly, solutions containing either 0.75% glutaraldehyde and 0.01% phthalaldehyde or


184 0.25% glutaraldehyde and 0.025% phthalaldehyde also are tuberculocidal within 30 minutes at 20° C. The tuberculocidal activity was not p H dependent (pH 3-9). The addition of 0.1% phthalaldehyde did not inhibit sporicidal activity of 2% glutaraldehyde.


Chemical Properties In contrast with the aliphatic glutaraldehyde (GTA), ortAo-phthalaldehyde is an aromatic dialdehyde compound with the formula CgH4(CHO)2 and a chemical (IUPAC) name of 1,2-benzenedicarboxaldehyde with the following structure (Figure 1): o

0 glutaraldehyde

^-phthalaldehyde

Figure 1. Comparison of glutaraldehyde and ortho-phthalaldehyde (1,2-benzenedicarboxaldehyde) chemical structures.

A commercially available OPA-based disinfectant is marketed as CIDEX®OPA Solution and contains 0.55% w/v orfAo-phthalaldehyde and was introduced into the market in 1999. Like glutaraldehyde, O P A is a dialdehyde. However, O P A is an aromatic compound, whereas glutaraldehyde is a straight chain hydrocarbon.

Mycobacteria Mycobacteria as a genus include many pathogens known to cause serious diseases in humans and animals, including tuberculosis and leprosy. Mycobacteria are generally classified into two categories based on growth rate: fast-growing species such as M. chelonae and M. smegmatis and slow-growing



185 species like M. tuberculosis and M. avium intercelluare. Tuberculosis (TB) is the world's leading cause of death that results from a single infections disease. T B affects an estimated 1.7 billion people, equal to almost one-third of the entire global population. Mycobacteria are widespread in the natural environment, typically living in water (including tap water treated with chlorine), food sources and decaying organic material. As a genus, they share characteristic cell walls that are thicker than those of many other bacteria and which are hydrophobic, waxy and rich in mycolic acids / mycolates. The mycobacterial cell wall is responsible in large part for the hardiness of this genus. Mycobacterial infections are notoriously difficult to treat and because of this unique cell wall can survive long exposure to acids, alkalis, and detergents. Since mycobacteria represent the most difficult to kill vegetative microorganisms, they are commonly used to measure the efficacy of disinfectants.

The Action of ^-Phthalaldehyde on Mycobacteria In contrast to chemotherapeutic agents, biocides have multiple targets within the microbial cell, and the overall damage to target sites results in the biocidal effect (4). artAo-Phthalaldehyde's primary mechanism for biocidal activity is the reaction with primary amino groups (Figure 2) found within multiple bacterial structures, and in particular with proteins of the cell wall (5). To reach proteins at the cell wall, antibacterial agents such as O P A must first diffuse through the mycobacteria's thick, lipid-rich protective coat, comprised of mycolic acids. Mycolic acids are long chain, beta-hydroxy-alpha-

OPA

Amino Acid (glycine)

Reaction Complex

Figure 2. Reaction of OPA with a primary amino acid (glycine), the primary mechanism by which OPA denatures critical mycobacteria cell wall proteins.



186 alkyl branched fatty acids and in mycobacteria are covalently linked to an arabinogalactan polymer. The bacterial cytoplasmic membrane is further protected by a peptidoglycan matrix linked to the arabinogalactan layer. This defensive structure featuring long chain fatty acids serves as a protective outer wall for the mycobacteria cells and can effectively exclude many types of biocides and antibacterial agents. However, lipophilic agents may utilize the lipid bilayer pathway to cross the cell wall, and this knowledge has driven the rational design of anti-tuberculosis drugs (6). In fact, "the lipophilic nature of the molecule will play a key role in the diffusion through hydrophobic domains such as biological membranes."(7) As such, the high lipophilicity of O P A is likely the predominant reason why the molecule exhibits much higher efficacy than other dialdehydes. To demonstrate the relationship between the lipophilicity of biocides and their efficacy against mycobacteria, an examination of cell surface hydrophobicity was performed using two strains of mycobacteria. In such an experiment, an aqueous suspension of M. chelonae was combined with an organic layer (hexadecane). After mixing and settling, the relative populations of the M. chelonae in each layer were examined. The degree to which cells were found to populate the organic layer as compared with the numbers remaining in the aqueous layer can provide some insight into their relative cell surface hydrophobicities (Figure 3). Mycobacteria cells that have a more hydrophobic outer coating should be found to a greater extent in the organic phase, as compared with a more hydrophilic species which would tend to remain in the aqueous phase. Two strains were chosen for testing: M chelonae A T C C 35752 was chosen to represent the "typical" mycobacteria, for comparison against a glutaraldehyde resistant strain of M. chelonae. Interestingly, the G T A resistant strain of M. chelonae .partitioned into the organic phase to an extent nearly three times greater than that of the less resistant strain (Figure 4), which clearly indicates a correlation between biocide resistance and the hydrophobicity of the cell wall. In a companion study, the mass fraction of a primary source of the cell surface hydrophobicity, fatty acid methyl esters, was compared for the two strains. The data (Table 1), indicated nearly three times the tuberculostearic acid in the GTA-resistant mycobacteria, a result consistent with the results of the partitioning experiment. However, a causative correlation is not entirely clear. Clearly the mechanism of glutaraldehyde resistance in GTA-resistant mycobacteria is related to its significantly higher cell surface hydrophobicity. The higher hydrophobicity tends to exclude molecules which are polar in nature, and to some extent also excludes glutaraldehyde, which has an octanol-water partition coefficient ( K ) of only 16. o-Phthalaldehyde, however, is a considerably less polar molecule ( K = 45), which is much more soluble in nonpolar phases (8). This solubility lends O P A its particular efficacy against oc

o c


187


hexadecane

Figure 3. Schematic diagram of cells originally found only in the aqueous phase partitioning between aqueous and organic phases. Cell Hydrophobicity Assay relates the probability of finding cells in the aqueous or organic phase to the cell surface hydrophobicity.

Figure 4. The relationship between cell hydrophobicity and glutaraldehyde resistance was demonstrated using a Cell Hydrophobicity Assay. GTA resistant M. chelonae partition into the organic phase to a far greater extent than sensitive M. chelonae which mostly remained in the aqueous phase.


188 Table I. Glutaraldehyde Resistant Mycobacteria: Cell Wall Fatty Acids


Fatty Acid Methyl Ester ( F A M E )

Mass Fraction F A M E M. chelonae M. chleonae ( A T C C 35752) (GTA Resistant)

n-hexadecanoate (palmitate)

7.9

19.1

D-10-methylstearic acid (tuberculostearic acid)

5.5

15.6

36.9

29.9

cis-9-octadecanoate (oleate)

mycobacteria as its compact, aromatic structure allows it to traverse the lipid layer that might exclude more polar compounds. In a subsequent experiment, GTA-resistant At. chelonae was exposed to both O P A and glutaraldehyde for a comparison of their relative efficacies. The data (Figure 5) clearly indicated that O P A ' s lipophilicity allowed for enhanced permeation across the mycobacteria's lipid layer, resulting in a total kill (6-log reduction) of bacteria in less than 15 minutes. Equivalent samples of mycobacteria treated with glutaraldehyde exhibited more than 100 survivors, even after 20 minutes. When compared with several other biocide molecules with lower lipophilicity including G T A , glyoxal, glyoxal and butanedial, 5-min kill data for M abscessus from Fraud et al. (9) indicated the clear superiority of O P A . Interestingly, when the calculated octanol water partition coefficients were compared with efficacy, a generally linear correlation was observed between the biocide efficacy and the ability to diffuse through hydrophobic domains. It should be noted that while lipid membranes are highly permeable to lipophilic molecules such as OPA, several additional factors can inhibit overall permeability of cell walls. For example, membrane fluidity decreases as the hydrocarbon chains lengthen and contain fewer cw-double bonds and ciscyclopropane groups. Also, drug resistant strains of M chelonae, M. fortuitum, M. smegmatis and M. phlei contain a larger fraction of a-mycolates with less permeable trans double bonds at the inner position (6). Glutaraldehyde-resistant M chelonae was found to not only exhibit increased cell surface hydrophobicity as above (10,11) but they also exhibited decreases in the monosaccharides of arabinogalactan (//). Finally, the protein cross-linking action of glutaraldehyde



189

I

1

1

1

I

1

0

5

10

15

20

25

Time (minutes) Figure 5. Comparison of relative activities of OPA and GTA for the inactivation of a GTA resistant strain of M. chelonae.

may ultimately inhibit its own uptake, whereas O P A is not an effective crosslinking agent, presumably due to the spatial relationship of its aldehyde groups.

Molecular Simulations of the Action of ^-phthalaldehyde While it is difficult to experimentally determine the individual contributions of molecular structure and lipophilicity to the overall efficacy of a biocide against mycobacteria, molecular dynamics simulations can provide some insight. In cooperation with Anton J. Hopfinger of the College of Pharmacy at the University of New Mexico, computer simulated mycobacterial lipid layers were challenged with models of OPA and glutaraldehyde. M O L S I M software was used to generate a total of 140,000 confirmations of the biocides in 0.001-ps intervals to determine overall permeability of the layer for each of the molecules. The results of molecular dynamics simulations (Figure 6) suggest that the enhanced efficacy of O P A over glutaraldehyde against mycobacteria could be almost entirely derived from the improved permeability afforded by O P A ' s lipophilic structure. The data suggested that given similar reactivities, biocides



190

Figure 6. MOLSIM screen shot, detailing the progression of unhydrated OPA through a simulated layer of mycolic acids.

that are compact and highly lipophilic will exhibit the greatest efficacy against mycobacteria, information usefiil in the rational design of biocide molecules.

The Equilibria of ^-phthalaldehyde A n additional complexity of O P A is the molecule's ability to undergo covalent hydration in an aqueous environment. In fact, because of the electronwithdrawing and "orf/io-effect" of the second carbonyl group, the equilibrium is shifted toward the hydrated form. A s the electron-withdrawing properties of the CH(OH)2 group are not as substantial as those of a C H O group, the addition of a second water molecule is not favored. Instead, an intramolecular nucleophilic attack of an oxygen atom of the CH(OH)2 group on the remaining carbonyl group occurs, resulting in a cyclic hemiacetal (72) (Figure 7). These equilibria potentially play a role in the inactivation of mycobacteria with O P A , as the hydration of O P A results in structures with octanol-water partition coefficients resembling that of glutaraldehyde. While in principal hydrated forms of O P A may react with nucleophiles, their ability to cross the mycobacteria lipid layer may be limited by their increased polarity.


191


Unhydrated O P A

Hydrated O P A

Figure 7. Comparison of the unhydrated OPA structure with the hydrated cyclic hemiacetal. In an aqueous biocide solution, about 10% exists in the unhydrated form while about 70% reacts with water to form the cyclic hemiacetal (13). About 20% exists as an acyclic monohydrate (not shown).

Conclusions It is generally thought that the primary action of o-phthalaldehyde on mycobacteria is the denaturing of critical proteins through the reaction with primary amino groups. The rate limiting step in the inactivation of mycobacteria appears to be the diffusion of biocides through the bacteria's waxy, outer coating. Biocides that have the highest lipophilicity appear to have the greatest efficacy, which is apparently related to their ability to permeate though mycolic acids and other hydrophobic regions of the outer cell wall. Glutaraldehyde resistant mycobacteria exhibit considerably higher cell surface hydrophobicity than other mycobacteria, which suggests that their outer structures effectively exclude more polar compounds such as G T A from sensitive, inner structures. The enhanced efficacy of O P A for the inactivation of glutaraldehyde resistant mycobacteria can be explained almost entirely by its significantly higher lipophilicity. Once biocide molecules such as O P A have traversed the waxy outer layers, from a strictly probabilistic viewpoint they will attack amino groups in the sequence as encountered by diffusion. Those amino groups found within the arabinogalactan-peptidoglycan complex, therefore, would likely be primary targets leading to the inactivation of the cell. Proteins of the cytoplasmic membrane would also be vulnerable to attack. While it is likely that O P A has intra-cellular targets, it is entirely possible that inactivation needs only to result from sufficiently denatured arabinogalactan, peptidoglycan and cytoplasmic membrane layers. O P A molecules which diffuse through the many cell wall


192 layers and enter the cytoplasm may attack intracellular targets. Though due to the lack of D N A mutagenicity, it is likely that the principal reactive sites for O P A lie within the arabinogalactan-peptidoglycan complex and cytoplasmic membrane layer. Cell penetration may not be necessary for efficacy. The predominant O P A species in an aqueous solution of O P A biocide is the hydrated cyclic hemiacetal. However, it is likely based on octanol water partition coefficients ( K ) and molecular dynamics simulations that the unhydrated O P A species will have the greatest propensity to permeate through mycobacteria's protective outer lipid layer. Hydrated OPA, glutaraldehyde, and other less lipophilic biocide molecules found on the outside of the cell wall will be less likely to permeate into the lipid layer. In effect, it is likely that 10% of an O P A biocide solution provides most of the mycobactericidal efficacy. Downloaded by COLUMBIA UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: September 7, 2007 | doi: 10.1021/bk-2007-0967.ch009

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13.

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ortho-Phthalaldehyde: Mechanisms of Action against Mycobacteria

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