Chlorhexidine
Vinod P. Menon
Chlorhexidine was first synthesized in 1950 in the laboratories of Imperial Chemical Industries Ltd (London, United Kingdom) during antimicrobial research into synthetic antimalarial agents of the proguanil type. It was found to possess a high level of antibacterial activity, low mammalian toxicity, and a strong affinity for binding to skin and mucous membranes. These properties led to the development of chlorhexidine principally as a topical antiseptic for application to such areas as skin, wounds, and mucous membranes and for dental use.1 It is used as a surgical hand scrub or rub, for preoperative bathing, nursery neonatal umbilical stump care, surgical site disinfection, and oropharyngeal decontamination in ventilator patients. It is widely used for skin antisepsis before placement of epidural, arterial, and central venous catheters (CVCs). Chlorhexidine has also been impregnated into medical materials, including vascular cannulas and dressings for both vascular and epidural catheters. It has been used for decades by dentists and oral surgeons to control gingivitis and periodontitis. In addition, chlorhexidine has been used as a pharmaceutical preservative, particularly in ophthalmic and surgical irrigation solutions, and as a disinfectant for items such as inanimate surfaces and instruments.2
Chlorhexidine can also be found in dressings, ointments, suppositories, and contraceptive gels, and it is available as an over-the-counter solution for disinfection of minor cuts and wounds. It acts as preservative agent in various liquid soaps, shower foams, cosmetics, toothpaste, lubricants, and medical ointments because it prevents bacterial growth.3 It is included as an antiseptic in the World Health Organization Essential Medicines list.
CHEMISTRYChlorhexidine is 1,6-di(4-chlorophenyl-diguanido) hexane, a bisbiguanide of the following formula:
Various resonance structures are assigned to the biguanide molecule. Although the biguanide structure shown previously—containing two iminic C=NH groups—is traditionally used for chlorhexidine and other biguanide structures, computational4 analyses have suggested that the most stable conformer is better represented by the structure without iminic C=NH groups but containing two C=NH2 primary amine sites.
Differences in the chemistry between the iminic groups of the nominal structure and the amine functionality may play an important role in understanding the degradation reactions of chlorhexidine.
Study of the related group of bisbiguanides has demonstrated that this compound, with a single chlorine substituent in each phenol ring, is the most active.5 Chlorhexidine itself is a strong base, practically insoluble in water (0.008% wt/vol at 20°C), that reacts with acids to form salts of the RX2 type. The water solubility of the different salts varies widely. Chlorhexidine was first made commercially available as the poorly soluble hydrochloride salt and subsequently as the moderately soluble acetate salt. The freely soluble gluconate salt was introduced in 1957 and is currently the favored form of chlorhexidine in disinfectant formulations.
Soluble chlorhexidine gluconate (CHG) cannot be easily isolated as a solid and is manufactured as a 20% wt/vol aqueous solution (eg, United States Pharmacopeia [USP] Chlorhexidine Gluconate Solution), higher concentrations being too viscous for convenient use. The diacetate salt has a solubility of 1.9% wt/vol (20°C), whereas the dihydrochloride and other inorganic salts are relatively insoluble (Table 22.1). The low solubility of the inorganic salts may cause problems of precipitation if a water-soluble salt such as the digluconate is formulated with, or diluted in, a solution containing inorganic anions such as chloride, sulfate, or nitrate. The aqueous gluconate salt solution is miscible with glacial acetic acid and with water and miscible with three times its volume of acetone and five times its volume of dehydrated alcohol; further addition of acetone or dehydrated alcohol yields a white turbidity.6 The CHG solution should not be added directly to neat alcohol because precipitation may occur. In addition to water, the gluconate salt is soluble in methanol.7 The gluconate salt is also soluble in hydrophobic vehicles with a hydrophilic-lipophilic balance (HLB) value less than 10, where the hydrophobic vehicles have two proximate hydrogen bonding groups with at least one group being a hydrogen donor. Examples include monoacylglycerides and higher alkanediols.8
Solutions and powders of chlorhexidine are colorless, or almost colorless, and usually odorless, although formulations prepared from the diacetate salt occasionally have an odor of acetic acid. Solutions prepared from all salts have an extremely bitter taste that must be masked in formulations intended for oral use. The taste quality-altering effects of chlorhexidine in humans were studied by analyzing confusions among taste stimuli in an identification task. Treatment with chlorhexidine was found to produce a profound and lengthy alteration of the salty taste of all salty compounds. It reduced the bitter taste of a subset of bitter compounds but had little effect on sweet and sour tastes.9
TABLE 22.1 Solubility of chlorhexidine base and salts in water at 20°C (% wt/vol) | ||||||||||||||||
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Chlorhexidine is moderately surface active and forms micelles in solution; the critical micellar concentration of the acetate is 0.01% wt/vol at 25°C.10 The unusually high solubility of the gluconate salt has been attributed to self-association and formation of large aggregates.11 Aqueous solutions of chlorhexidine are most stable within the pH range 5 to 8. Above pH 8.0, chlorhexidine base is precipitated, and in more acid conditions, there is gradual deterioration of activity because the compound is less stable. Hydrolysis yields para-chloroaniline; the amount is insignificant at room temperature, but it is increased by heating above 100°C, especially at alkaline pH.12
Chemical analysis of chlorhexidine, its degradation products, and its impurities can be performed using a variety of different methods. Qualitative assays for identification of the various chlorhexidine reagents are reported in the respective USP and European Pharmacopoeia monographs.13,14 These may include infrared (IR) and melting point analyses. Quantitative analyses can generally be performed by several nonseparation methods including UV absorbance, acid titrimetry, and gravimetric determination of insoluble copper complexes in pharmaceutical products with chlorhexidine. High-performance liquid chromatography (HPLC) is the method most used to analyze this antiseptic; solid-phase extraction with UV spectrophotometry, gas-liquid chromatography, liquid chromatography, capillary electrophoresis, flow injection extraction-spectrophotometry, and voltammetry also are used to assess it.15 Some induced colorimetric determinations of biguanides have been suggested.16 Thin-layer chromatography methods have been used to evaluate CHG solutions, but although rapid, these lack the quantitative rigor and resolving power of HPLC. Para-chloroaniline in its free base form can be analyzed by gas chromatography (GC) methods; however, care must be exercised to avoid artificial increases in its concentration through thermal degradation of chlorhexidine during the GC injection process. Prolonged exposure to light should also be avoided.
Chlorhexidine Impurities and Degradants
Despite the widespread use of chlorhexidine in products, relatively little has been published with respect to the nature and characterization of the impurities present in chlorhexidine preparations. This dearth of data exists
despite well-known issues with the degradation of chlorhexidine accelerated by thermal, acid/base, and photolytic processes. The most extensive characterization of the impurities and degradation products of chlorhexidine was published by Revelle et al from the US Food and Drug Administration (FDA).17,18 The Revelle group identified, and confirmed by synthesis, 11 common impurities. The group encountered general retro-ene degradation of biguanide groups to guanide products, and aqueous acid catalyzed conversion of guanide groups to ureas, then to carbamates, and finally to amines. Although thermal and acidic stresses degraded chlorhexidine to similar products, dechlorinated degradation products were uniquely obtained through photolytic reactions. Ha and Cheung19 identified the same products formed by an alternate hydrolytic chemical approach. The European Pharmacopoeia14 identifies 12 impurities without further attribution or explanation.
despite well-known issues with the degradation of chlorhexidine accelerated by thermal, acid/base, and photolytic processes. The most extensive characterization of the impurities and degradation products of chlorhexidine was published by Revelle et al from the US Food and Drug Administration (FDA).17,18 The Revelle group identified, and confirmed by synthesis, 11 common impurities. The group encountered general retro-ene degradation of biguanide groups to guanide products, and aqueous acid catalyzed conversion of guanide groups to ureas, then to carbamates, and finally to amines. Although thermal and acidic stresses degraded chlorhexidine to similar products, dechlorinated degradation products were uniquely obtained through photolytic reactions. Ha and Cheung19 identified the same products formed by an alternate hydrolytic chemical approach. The European Pharmacopoeia14 identifies 12 impurities without further attribution or explanation.
Classes of chlorhexidine-related impurities and degradation products found in aged CHG-containing products tend to arise from three major sources or mechanisms:
Manufacturing impurities: Side products from the manufacture of the chlorhexidine-free base form are carried over into the subsequent chlorhexidine salts. These include species such as the mono-ortho-chloro isomer resulting from low-level ortho-chloroaniline impurities in the para-chloroaniline synthetic reagents. The levels of these species do not generally change significantly with sample aging.
Chlorhexidine degradation reactions: Chlorhexidine and its salts can degrade by various homogeneous and heterogeneous reactions. In general, the rates of these degradation reactions are significantly higher in solution than in solid reagent forms. These degradation reactions proceed primarily through three mechanisms: retro-ene reactions, hydrolysis reactions, and reactions with the counterion in the chlorhexidine salt.
Side reactions with formulation reagents or sterilization byproducts: Reactions of biguanide species with aldehydes, such as acetaldehyde, present at low levels in formulations including CHG can lead to systematic patterns of new impurities, which grow slowly with time. Free radical generators, including gamma irradiation used to treat such products, can result in formation of dechlorination products.
PHARMACEUTICAL ASPECTSCompatibility
Chlorhexidine is a cationic molecule in its salt forms and is thus generally compatible with other cationic materials, such as quaternary ammonium compounds (eg, cetrimide, benzalkonium chloride), although compatibility will depend on the nature and relative concentration of the second cationic species. It is, however, possible for a reaction to occur between chlorhexidine and the counterion of a cationic molecule, resulting in the formulation of a less soluble chlorhexidine salt, which may then precipitate.
Nonionic substances, although not directly incompatible with chlorhexidine salts, may inactivate the antiseptic to varying degrees, according to the chemical type and concentration used. In many cases, a suitable ratio of chlorhexidine to excipient can be chosen to give the required degree of bioavailability and hence activity, and this should be confirmed by suitable microbiologic tests. Chlorhexidine salts are compatible with most cationic and nonionic surfactants, but at higher concentrations of surfactant, chlorhexidine activity can be substantially reduced owing to micellar binding. In hard water, insoluble salts may form owing to interaction with calcium and magnesium salts; aqueous dilution is preferably carried out with deionized water. Solubility may be enhanced by the inclusion of surfactants such as cetrimide.20
Chlorhexidine is incompatible with inorganic anions in all but extremely dilute solutions (see Table 22.1). This incompatibility sometimes may be overcome by adding a suitable solubilizing agent in formulations in which this is acceptable. Residual antimicrobial activity of chlorhexidine on the skin can be reduced after a saline rinse or soak.21 Chlorhexidine is also incompatible with organic anions, such as soaps, sodium lauryl sulfate, sodium carboxymethyl cellulose, alginates, and many pharmaceutical dyes. Commonly used synthetic anionic thickening agents in hydroalcoholic hand sanitizers, like carbomer and acrylates/C10-30 alkyl acrylate crosspolymer, have also been shown to have a significant negative impact on the persistent activity of chlorhexidine on the skin.22 In certain instances, there will be no visible signs of incompatibility, but the antimicrobial activity may be significantly reduced because of the chlorhexidine being incorporated into micelles. Chlorhexidine forms inclusion complexes with β-cyclodextrins that can modulate both its efficacy and mammalian cytotoxicity.23
Effect of pH on Activity
The antimicrobial activity of chlorhexidine is pH dependent; the optimum range of 5.5 to 7.0 corresponds to the pH of the body surfaces and tissues. Within the pH range 5 to 8, antibacterial activity will vary with the microorganism and the type of buffer used. The pH in chronic wounds most commonly has a range of 6.5 to 8.5.24 Notably, in vitro testing of activity against Staphylococcus aureus and Pseudomonas aeruginosa, among the most prominent bacteria in wound infections, showed pH independence in the range of 5.0 to 9.0.25
Isotonicity
Dilution of chlorhexidine in physiologic saline to render it isotonic with plasma should be regarded with caution because of the low solubility of chlorhexidine hydrochloride (<1 mg/100 mL). Although solutions may be free of precipitate on preparation, the solutions (normally containing at least 0.02% chlorhexidine) will be supersaturated, and precipitation of the hydrochloride salt is likely to occur on standing.
Sodium acetate may be used to adjust the tonicity of chlorhexidine solutions without the problem of precipitation; however, the pH of the required solution (2.1% wt/vol sodium acetate, European Pharmacopoeia) may be as high as 8.0 and should not, therefore, be stored for prolonged periods.
Coloring Agents
Only a limited number of approved dyes can be used to color chlorhexidine solutions, and even these are anionic in nature and therefore may not be fully compatible. They usually can be added at low concentrations to tint chlorhexidine solutions for identification purposes but are likely to form a precipitate when used at the higher concentrations necessary to give good skin-staining properties. For example, carmoisine (E122) at a concentration of 0.0005% provides sufficient coloring for identification purposes and will remain stable for long periods. At a concentration of 0.05%, it has good skin-staining properties but is usually given a shelf life of 7 days after preparation. On the other hand, there are commercial shelf-stable hydroalcoholic CHG skin preparation compositions with skin-staining properties for visualization of the prepped field that are tinted with approved dyes.26 The stability of the tinted active is thus dependent on the solution composition as well as the identity, composition, and concentration of the individual tints. Solution tinting prevents inadvertently confusing the clear antiseptic with other clear medical solutions such as an injectable active drug solution. Such confusion can lead to tragic results.27 Additionally, selection of the appropriate tint is important in applications such as preoperative skin preparations, where skin pigmentation can influence visibility. Failure to determine the field of prepared skin adequately could lead to an increased risk of surgical site infection (SSI). The tint enables surgeons to visualize the preparation of the skin adequately, which is a crucial component of anti-infection precautions.28
Packaging
The nature and quality of containers for concentrates and use dilutions are important. Glass, high-density polypropylene, and high-density polyethylene are usually suitable. Low-density polyethylene may be unsuitable because of excessive adsorption, and other packaging materials may interact with the antiseptic. Cork stoppers or cap linings never should be used because water-soluble tannins present may inactivate chlorhexidine.29 Solutions of chlorhexidine should also be protected from prolonged exposure to light.
Sterilization
Dilute solutions of chlorhexidine (<1.0% wt/vol) may be sterilized by steam at 115°C for 30 minutes or at 121°C to 123°C for 15 minutes. Autoclaving of solutions greater than 1.0% can result in the formation of insoluble residues and is therefore unsuitable. Following autoclaving of a 0.02% wt/vol CHG solution at pH 9 for 30 minutes at 120°C, it was found that 1.56% wt/wt of the original chlorhexidine content had been converted into para-chloroaniline; for solutions at pH 6.3 and 4.7, the para-chloroaniline content was 0.27% wt/wt and 0.13% wt/wt, respectively, of the original gluconate content.30 If sterile solutions are required at such high concentrations, filtration through a 0.22 µm membrane filter is recommended; however, the first 10 mL should be discarded because adsorption to the filter can occur in the initial stage; fibrous and porcelain filters are unsuitable.
Chlorhexidine hydrochloride powder is stable to dryheat sterilization at 150°C. The solid salts are stable to sterilizing doses of gamma radiation, but chlorhexidine in solution is decomposed. Electron beam sterilization of a CHG/ethanol disinfectant combination results in more than 20% of the chlorhexidine content being altered by irradiation at the sterilization dose.31
Unsterilized aqueous chlorhexidine solutions or devices can be inadvertently contaminated by certain tolerant bacilli such as the Burkholderia cepacia complex or Serratia marcescens, which can lead to the antiseptic itself becoming the nidus of an infection outbreak.32 Medical devices impregnated with chlorhexidine are routinely safely sterilized using the ethylene oxide modality without altering the activity of the drug or introducing harmful levels of residuals.33,34,35
Storage
Dilute chlorhexidine solutions may be stored at room temperature, and a shelf life of at least 1 year can be expected, provided preparation and packaging are adequate. Prolonged exposure to high temperature or light is to be avoided because this can adversely affect stability; chlorhexidine solutions exposed to light become discolored due to the polymerization of para-chloroaniline.12,36 All dilute solutions to be stored should be either heattreated (sterilized or pasteurized) or chemically preserved
(4% isopropanol or 7% ethanol) to eliminate the possibility of microbial contamination. For autoclaved solutions, the choice of container material is important, the best results being achieved by using neutral glass or polypropylene. Guidelines on the storage of locally prepared aqueous solutions are as follows, but care should be taken to review the antiseptic formulation instructions for use and safety sheets:
(4% isopropanol or 7% ethanol) to eliminate the possibility of microbial contamination. For autoclaved solutions, the choice of container material is important, the best results being achieved by using neutral glass or polypropylene. Guidelines on the storage of locally prepared aqueous solutions are as follows, but care should be taken to review the antiseptic formulation instructions for use and safety sheets:
Untreated solutions: Prepare and use within 24 hours.
Do not store.
Chemically preserved solutions: Store unopened for a maximum of 3 months. When opened, use within 7 days.
Sterilized solutions: Store unopened for a maximum of 12 months. When opened, use within 24 hours.
Chlorhexidine and Laundering
Chlorhexidine is absorbed onto the fibers of certain fabrics, particularly cotton, and resists removal by washing. If a hypochlorite (chlorine-releasing) bleach is used during the washing procedure, a fast brown stain may develop because of a chemical reaction between the chlorhexidine and hypochlorite causing precipitate formation.
There has been considerable debate about the composition of the precipitate, with different analytical techniques showing contradictory results and some indicating the presence of para-chloroaniline.37 A decisive study employing a variety of techniques including high-performance, thin-layer, and GC techniques, proton nuclear magnetic resonance (1H NMR), IR spectroscopy, and GC/mass spectroscopy (MS) showed that para-chloroaniline was not typically present in the precipitate.38 Chlorophenylurea has been proposed as one of the components of the precipitate.39 This problem of staining can be avoided by eliminating the use of bleach or replacing the chlorine-releasing bleach with one that is peroxide based, such as sodium perborate. Pretreatment of the fabrics with a dilute acidic detergent reduces or eliminates staining when a chlorine bleach is subsequently used.40
MICROBIOLOGYThe antimicrobial activity of chlorhexidine is directed mainly toward vegetative gram-positive and gramnegative bacteria; it is inactive against bacterial spores except at elevated temperatures, and acid-fast bacilli are inhibited but not killed by aqueous solutions. The infectivity of some lipophilic viruses (eg, influenza virus, herpes virus, human immunodeficiency virus [HIV]) is rapidly inactivated by chlorhexidine, although aqueous solutions are not active against the small nonlipid viruses. Yeasts (including Candida albicans) and dermatophytes are usually sensitive, although chlorhexidine’s fungicidal action in general is subject to species variation, as is observed with other biocides.
Mechanisms of Antibacterial Action
The mechanism of action of chlorhexidine and related biguanides was reviewed by Woodcock.41 At relatively low concentrations, the action of chlorhexidine is bacteriostatic, and at higher concentrations, it is rapidly bactericidal, with the actual levels varying somewhat from species to species.
The lethal process consists of a series of related cytologic and physiologic changes, some of which are reversible, that culminate in the death of the cell. The sequence is thought to be as follows: (1) rapid attraction toward the bacterial cell; (2) specific and strong adsorption to certain phosphate-containing compounds on the bacterial surface; (3) overcoming the bacterial cell wall exclusion mechanisms; (4) attraction toward the cytoplasmic membrane; (5) leakage of low-molecular-weight cytoplasmic components, such as potassium ions, and inhibition of certain membrane-bound enzymes, such as adenosyl triphosphatase; and (6) precipitation of the cytoplasm by the formation of complexes with phosphated entities, such as adenosine triphosphate and nucleic acids.
Characteristically, a bacterial cell is negatively charged, and the nature of the ionogenic groups varies with bacterial species. It has been shown that given sufficient chlorhexidine, the surface charge of the bacterial cell is rapidly neutralized and then reversed. The degree of charge reversal is proportional to the chlorhexidine concentration and was found to reach a stable equilibrium within 5 minutes. The rapid electrostatic attraction of the cationic chlorhexidine molecules and the negatively charged bacterial cell undoubtedly contributes to the rapid rate of kill associated with chlorhexidine, although surface charge reversal is secondary to cell death. Electron microscopy and assays for characteristic outer membrane components, such as 2-keto-3-deoxyoctonate (KDO), demonstrate that sublethal concentrations of chlorhexidine bring about changes in the outer membrane integrity of gram-negative cells. An efflux of divalent cations, especially calcium ions, occurs prior to or during such outer-membrane changes. Chlorhexidine molecules are thought to compete for the negative sites on the peptidoglycan, thereby displacing metallic cations. Being 6 carbons long, rather than 12 to 16 carbons, the hydrophobic inner functionality of the chlorhexidine molecule is somewhat inflexible and incapable of folding sufficiently to interdigitate into the cell bilayer. Chlorhexidine therefore bridges between pairs of adjacent phospholipid head groups each being bound to a biguanide moiety and displaces the associated divalent cations. Interestingly, the distance between phospholipid headgroups in a closely packed monolayer is roughly equivalent to the length of a hexamethylene grouping.
A bisbiguanide would therefore be capable of binding to two adjacent phospholipid head groups. Such binding is critical for the bisbiguanides as activity is reduced significantly if the polymethylene bridge is made longer or shorter than six carbons. Although the action of multidrug efflux pumps is able to moderate the efficacy of quaternary ammonium compounds at low concentrations, they have relatively little effect42 on the action of bisbiguanides. This is presumably because the bisbiguanides do not become solubilized within the membrane core.43
A bisbiguanide would therefore be capable of binding to two adjacent phospholipid head groups. Such binding is critical for the bisbiguanides as activity is reduced significantly if the polymethylene bridge is made longer or shorter than six carbons. Although the action of multidrug efflux pumps is able to moderate the efficacy of quaternary ammonium compounds at low concentrations, they have relatively little effect42 on the action of bisbiguanides. This is presumably because the bisbiguanides do not become solubilized within the membrane core.43
In terms of the lethal sequence, the bacterial cytoplasmic membrane appears to be the important site of action. Several changes indicative of damage to the cytoplasmic membrane have been observed in bacterial populations treated with bacteriostatic and bactericidal levels of chlorhexidine. Leakage of cytoplasmic contents is a classic indication of damage to the cytoplasmic membrane, starting with low-molecular-weight molecules typified by potassium ions. Electron micrographs of these sublethally treated cells (Figure 22.1) show a shrinkage or plasmolysis of the protoplast.44 Cells treated with bacteriostatic levels of compound can recover viability despite having lost up to 50% of their K+. This is particularly true if the excess chlorhexidine is removed by a neutralizing agent, as happens in many in vitro testing situations.
 FIGURE 22.1 Cytological changes of Escherichia coli after treatment with chlorhexidine. Control cells possess intact cell membrane, cell wall, and complete cell content (A). After 4 h of incubation with 0.75 mg/L chlorhexidine, the treated E coli cells showed detached cytoplasmic membrane from the cell wall at both poles and cylindrical part of the cells (see arrows in B), leakage of cell content (see arrow in C), and formation of ghost cells (D). Magnification in all cases = ×20 500. Bar = 1 µm. Reproduced from Cheung et al.44 |
As the chlorhexidine concentration is increased, higher molecular weight cell contents, such as nucleotides, appear in the supernatant fluid around the cell. Bacterial cells showing more than a 15% increase in nucleotide leakage have been found to be damaged irreversibly; levels of chlorhexidine producing this effect are therefore
considered bactericidal. The rate of membrane disruption and cell leakage increases with chlorhexidine concentration up to a maximum and then falls back, and at concentrations that are rapidly bactericidal (100-500 mg/L), release of cell components does not occur. Electron microscopy shows the cytoplasm of these cells to be chemically precipitated—this precipitation having been caused by an interaction between the chlorhexidine and phosphated entities within the cytoplasm, such as adenosine triphosphate and nucleic acids.44
considered bactericidal. The rate of membrane disruption and cell leakage increases with chlorhexidine concentration up to a maximum and then falls back, and at concentrations that are rapidly bactericidal (100-500 mg/L), release of cell components does not occur. Electron microscopy shows the cytoplasm of these cells to be chemically precipitated—this precipitation having been caused by an interaction between the chlorhexidine and phosphated entities within the cytoplasm, such as adenosine triphosphate and nucleic acids.44
Antimicrobial Spectrum
Although numerous publications refer to the bacteriostatic and bactericidal properties of chlorhexidine against particular microorganisms, the methods used vary, and it is often difficult to compare results. A series of studies were therefore performed to provide a comprehensive spectrum of activity for chlorhexidine using both microbiostatic and microbicidal methods. The strains of organisms tested include clinical isolates, laboratory strains, and standard culture collection types. Each strain was tested to determine the minimum inhibitory concentration (MIC) of chlorhexidine and its susceptibility to the bactericidal action of 0.05% aqueous CHG using a rate-of-kill method.
Minimum Inhibitory Concentration Method
Twofold dilutions of CHG were prepared in Iso-Sensitest Agar, the surface of which was inoculated with a suspension of each test organism. After incubation at 37°C for 24 hours, the agar was examined for distinct growth. The MIC was recorded as the lowest chlorhexidine concentration that prevented growth.
Molds and yeasts were tested on Sabouraud agar incubated at 30°C for 24 to 72 hours. Anaerobes were incubated anaerobically for 2 to 3 days on agar containing 5% lysed blood. Fastidious organisms were incubated in carbon dioxide for 2 to 3 days (Tables 22.2 and 22.3).
Rate-of-Kill Test
The in vitro bactericidal and fungicidal activity of 0.05% CHG was determined using a procedure based on BS EN 1276:2009.45 One milliliter of a 24-hour broth culture of the test organism was added to 10 mL of aqueous 0.05% wt/vol CHG solution, which was maintained at ambient temperature (18°C-21°C). One-milliliter aliquots of the mixture were removed after 20 seconds, 1 minute, and 10 minutes and transferred to inactivator broth containing 1.5% soya lecithin and 10% polysorbate 80. A viable count was performed on appropriate further dilutions, and by comparison with an untreated control, a 10 log reduction factor was calculated (Tables 22.4 and 22.5).
Bacterial Susceptibility
The susceptibility of individual bacterial strains to chlorhexidine varies widely; however, few have been found to be capable of surviving concentrations of the antiseptic encountered in use.46,47 It has been suggested that prolonged use of the antiseptic may lead to reduced susceptibility and to the development of resistant bacteria; however, this is not supported by the work of Martin48 and Simpson et al,49 who found that bacterial strains encountered in areas of prolonged and extensive use of the antiseptic have similar susceptibilities to strains of the same species encountered in areas where there is little or no chlorhexidine.
There is also no good evidence that the plasmidmediated antibiotic resistance common among gramnegative bacteria is associated with resistance to chlorhexidine. Michel-Briand et al,50 Ahonkhai et al,51 and Sykes and Matthew52 were unable to find any increase in chlorhexidine-resistance among antibiotic-resistant strains of Escherichia coli, P aeruginosa, S marcescens, or Proteus mirabilis.
Early studies with strains of methicillin-resistant S aureus (MRSA) using bacteriostatic MIC test procedures demonstrated a degree of reduced sensitivity to chlorhexidine compared to methicillin-sensitive strains of this organism (methicillin-sensitive S aureus [MSSA])53,54; however, this is considered of little clinical relevance because the highest MIC value for chlorhexidine quoted in these studies is 4 mg/L, and therefore, all strains of S aureus, including MRSA, can be regarded as sensitive to user concentrations of chlorhexidine. Both Haley et al55 and Cookson et al56 found the bactericidal activity of a 4% chlorhexidine hand wash to be similar for strains of MRSA and MSSA. A number of clinical reports also support the use of chlorhexidine preparations as part of programs for the control of outbreaks with MRSA57,58,59; however, Kampf et al60 found a chlorhexidine in alcohol hand rub to be more effective than a chlorhexidine-based hand wash against MRSA and recommend this form of hand disinfection by staff treating MRSA patients.
Several outbreaks associated with contaminated CHG solutions have been reported,61 indicating the ability of microorganisms to adapt to CHG. One mechanism of CHG resistance is through the cellular expression of efflux pumps, which can pump out of the bacterial cell various types of antibiotics and biocidal agents including CHG.62 An indication of their ability can be gleaned through the study of 114 effluxing S aureus isolates where CHG was effluxed in 96% of the strains.63 Other mechanisms of resistance can include the inactivation of the active ingredient or changes in the cell wall structure.64
TABLE 22.2 Bacteriostatic activity of chlorhexidine gluconate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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The MIC and minimum bactericidal concentration (MBC) are commonly used to detect reduced susceptibility to chlorhexidine. However, there is neither a defined standardized method nor a consensus on the meaning of resistance to this agent.65 Recently, Morrissey et al66 attempted to define break points for chlorhexidine on the basis of normal distribution of MICs for a given bacterial species, known as the epidemiologic cutoff value. This value is described as the upper limit of the normal MIC distribution for chlorhexidine for a specific species and not the likelihood of treatment failure. In general, the advised dose of chlorhexidine usage is several times higher than the MBC; yet, if chlorhexidine concentration reaches sublethal levels over time,67 those isolates with reduced susceptibility to chlorhexidine will remain viable, survive, and possibly persist. A more recent study68 identifies the characteristics of lineages of S aureus with reduced susceptibility to chlorhexidine. The conclusion from this study is that clinical isolates with reduced susceptibility to chlorhexidine consist of strains that are genetically heterogeneous in their possession of biocide resistance genes. In order to reduce selection pressure in nosocomial pathogens, it has been suggested that the use of CHG be restricted to those indications with a clear patient benefit and to eliminate it from applications with a doubtful benefit.69 Still, the overall risk for an acquired resistance to CHG is considered to be small as long as the antiseptics are used correctly.
TABLE 22.3 Fungistatic activity of chlorhexidine | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Virulence Suppression
Whereas killing potentially pathogenic bacteria certainly will prevent them from causing infection, certain types of sublethal chemical treatment might also alter or damage bacterial cells in such a way as to reduce their ability to initiate the disease process. Thus, the bacteria could still be viable but less pathogenic. The ability of chlorhexidine to produce such a “depathogenizing” effect was first investigated by Holloway et al70 using a peritonitis model in mice. Pathogenic strains of E coli and Klebsiella aerogenes were treated with sublethal concentrations of chlorhexidine after which the antiseptic was neutralized and the test suspension injected into susceptible animals. The results of these studies demonstrated that the pathogenicity of bacteria surviving treatment with chlorhexidine was reduced by more than 90%. This was confirmed by Rotter et al,71 who could not demonstrate a similar effect with alcohol. This effect of chlorhexidine must be considered secondary to the direct bactericidal activity of the antiseptic; however, it is believed to be an additional, clinically relevant property that is not evident in conventional in vitro studies concerned with viability alone.
Sporicidal Activity
Chlorhexidine will inhibit the growth of the vegetative cells of spore-forming bacteria at relatively low concentrations (see Table 22.2) and also will inhibit spore germination/outgrowth. It is generally recognized, however, that chlorhexidine has little direct sporicidal activity except at elevated temperatures. Shaker et al72 investigated the sporicidal activity of an aqueous solution of CHG (25 mg/L) against Bacillus subtilis spores at various temperatures. At 20°C, 30°C, and 37°C, the antiseptic had little effect on spore viability, even after 120 minutes exposure. At a temperature of 70°C, however, the antiseptic reduced the number of spores by 5 logarithms. Physical and chemical conditions that alter the protective barriers of Clostridium difficile spores convey sporicidal activity to chlorhexidine. The C difficile spores became susceptible to heat killing at 80°C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water, which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04% to 0.0004% wt/vol chlorhexidine solutions at 80°C. Reduction of spores exposed to 4% wt/vol chlorhexidine solutions at moderate temperatures (37°C and 55°C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55°C. Elevating the pH to 9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions.73
Virucidal Activity
Chlorhexidine has good activity against viruses with a lipid component in their coats or with an outer envelope.
These include many respiratory viruses, herpes, and cytomegalovirus. The in vitro bactericidal and virucidal activity of throat lozenges containing CHG in relation to the main microorganisms responsible for upper respiratory tract infections, including the H1N1 influenza virus, was evaluated after short (5 min) and long (3 h) contact times. Antiviral activity inducing a 2 log (99%) destruction of the H1N1 virus after a 5-minute contact time at high CHG concentration was noted.74
These include many respiratory viruses, herpes, and cytomegalovirus. The in vitro bactericidal and virucidal activity of throat lozenges containing CHG in relation to the main microorganisms responsible for upper respiratory tract infections, including the H1N1 influenza virus, was evaluated after short (5 min) and long (3 h) contact times. Antiviral activity inducing a 2 log (99%) destruction of the H1N1 virus after a 5-minute contact time at high CHG concentration was noted.74
TABLE 22.4 Bactericidal activity of 0.05% chlorhexidine gluconate | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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In common with many other antiseptics, however, aqueous solutions of chlorhexidine do not have any significant activity against the small nonenveloped viruses, which include many of the enteric viruses, poliomyelitis, and papilloma (warts) virus.75
The HIV, the organism responsible for acquired immunodeficiency syndrome (AIDS), is known to be one of the enveloped viruses and can, therefore, be predicted to be sensitive to the action of chlorhexidine. This was confirmed in a series of in vitro studies. A 4% chlorhexidine hand wash preparation and 0.5% chlorhexidine in 70% alcohol were both found to be 100% effective against HIV type I after a 15-second contact. Aqueous solutions of chlorhexidine down to a final test concentration of 0.05% were 100% effective within 1 minute.76 In a separate series of studies, chlorhexidine at 1 mg/mL (0.1%) was 80% to 100% effective.77 A study in 2001 showed that intrapartum chlorhexidine lavage is not effective at preventing mother-to-child HIV transmission when used intravaginally
during delivery. However, lavage with 0.4% chlorhexidine solely before rupture of the membranes tended toward lower transmission rates.78 Published data on the activity of chlorhexidine against a wide range of viral agents are summarized in Table 22.6.75,79,80,81,82,83,84,85,86,87,88
during delivery. However, lavage with 0.4% chlorhexidine solely before rupture of the membranes tended toward lower transmission rates.78 Published data on the activity of chlorhexidine against a wide range of viral agents are summarized in Table 22.6.75,79,80,81,82,83,84,85,86,87,88
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