The Rotation of Disinfectants Principle: True or False?

A disinfectant is a chemical agent that kills microorganisms on inanimate objects and surfaces. It is similar to an antiseptic, except that antiseptics are used on living tissue. Some disinfectants are also antiseptics and vice versa. Disinfectants do not typically kill bacterial spores, although some disinfectants such as hydrogen peroxide (H₂O₂) and chlorine can kill bacterial spores when applied in high concentrations.

(LIANE RISS/GETTY IMAGES)

Disinfectants are biocidals, and biocidal activity is measured by the minimum bactericidal concentration (MBC). When a microorganism is first exposed to a disinfectant and subculturing is not possible, it is deemed to have been killed. The concentration at which the microorganism is killed is known as biocidal activity. The effects of antibiotics, such as penicillin and cephalosporin, are also described in terms of biocidal activity (1).

Several forums and publications have claimed that disinfectants must be rotated in biotechnology and pharmaceutical manufacturing settings to prevent the target organisms from developing resistance. Chapter <1072>, "Selection of a Disinfectant for Use in a Pharmaceutical Manufacturing Environment," in USP 30 addresses disinfectant rotation. It states that:

The development of microbial resistance to antibiotics is a well-described phenomenon. The development of microbial resistance to disinfectants is less likely, as disinfectants are more powerful biocidal agents than antibiotics and are applied in high concentrations against low populations of microorganisms usually not growing actively, so the selective pressure for the development of resistance is less profound. However, the most frequently isolated microorganisms from an environmental monitoring program may be periodically subjected to use dilution testing with the agents used in the disinfection program to confirm their susceptibility.

The Japanese Pharmacopoeia, British Pharmacopoeia, and European Pharmacopoeia do not currently address the issue of disinfectant rotation.

Annex 1 of the European Commission's Good Manufacturing Practice (GMP) Guidelines, "Manufacture of Sterile Medicinal Products" states, "Where disinfectants are used, more than one type should be employed. Monitoring should be undertaken regularly in order to detect the development of resistant strains" (2).

But, the US Food and Drug Administration does not mention the rotation of disinfectants in its equivalent guideline Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice (3). This discrepancy raises the question of whether the technique of rotating disinfectants has a sound scientific rationale.

Correct terms

Many people use inaccurate expressions when they refer to disinfectants. For example, it is common to confuse sanitizers with disinfectants. Although these agents are similar, they are not the same. Disinfection targets pathogenic microorganisms, and sanitation kills all microorganisms. Table I presents a glossary of terms, as published by Gilbert and McBain and FDA, to dispel confusion (1, 4).

Table I: Glossary of terms.

The biotechnology and pharmaceutical industries usually clean equipment with a detergent and use a disinfectant to reduce or eliminate microbial contamination. These industries usually call the process of reducing microbial contamination "disinfection," but the correct term is "sanitation" or "sanitization." As a side note, the probability of finding a true pathogen in an environment that complies with current good manufacturing practice (CGMP) is quite low.

This article uses the following definitions, which are described in references 5 and 6:

• Resistance is the property of a microorganism that can survive but not grow, either alive or in stasis, at the recommended exposure conditions and use concentration of a disinfectant.

• Tolerance is the relative capacity of a microorganism to survive and grow at or beyond the recommended exposure conditions and use concentration of a disinfectant.

• Susceptibility is the failure of a microorganism to survive exposure to a disinfectant at the recommended conditions and concentration.

• Reduced susceptibility is the property of a microorganism that is not killed by the usual disinfectant concentration and exposure conditions, but does not survive a higher concentration or different conditions.

Clinical microbiologists use susceptibility tests to predict the probability of an antibiotic's therapeutic success. To say that bacteria are susceptible to an antibiotic means that, under laboratory conditions, the minimum inhibitory concentration (MIC) of that antibiotic complied with the recommended standard for the target microorganism. But therapeutic success is not assured because the interactions between the host and the microorganism also influence therapeutic success.

Resistance occurs when the required amount of antibiotic exceeds the minimum MIC and the likelihood of therapeutic success is low. Reduced susceptibility to an antibiotic occurs when the recommended concentration to achieve therapeutic success is not effective and a higher concentration is required to achieve therapeutic success. When an antibiotic complies with the minimum MIC but is ineffective against an infection by a microorganism, the microorganism is said to be resistant, regardless of whether the failure was caused by the host, antibiotic, or clinician.

Classes of disinfectants and examples of resistance

Studies of disinfectants' mechanisms of action generally indicate that, unlike antibiotics, whose high levels of target-specificity facilitate selective action against specific cell targets, disinfectants work at many sites within the cell. The damage caused by disinfectants is seldom accomplished through a single injury and is a direct consequence of exposure to or chemical attack by the disinfectant's active ingredients. That is, the actions of disinfectants are, in most cases, not pharmacologically precise (1). In the case of bacteriostatic antibiotics, the host's immune response plays an important role in eradicating the invading microorganisms. Disinfectant formulations contain biocidal chemicals at concentrations high enough to affect multiple rather than unique targets. Supplementary ingredients are also included in disinfectant formulations to enhance the killing effects.

The activity of disinfectants is measured by MIC or MBC. In this sense, the antibacterial ingredient could be an antibiotic, an antiseptic, or a disinfectant. The MIC is determined by visual inspection of the media used in the test, and the MBC requires plate counts to quantify the amount of inactivated or killed microorganisms.

Disinfectants are classified in two broad categories: oxidizing disinfectants and nonoxidizing disinfectants. These two categories are further subdivided. Tables II and III list common disinfectants (and some antiseptics) and their modes of action. These lists are not exhaustive; only the most relevant examples are presented.

Table II: Oxidizing disinfectants.

Oxidizing agents

As Tables II and III show, most disinfectants' mechanism of action is nonspecific and damages cells at different targets. The most effective disinfectants are oxidizing agents, followed by reducing agents.

Table III: Nonoxidizing disinfectants.

Hydrogen peroxide. Helicobacter pylori has shown resistance to hydrogen peroxide at a use concentration of ~3% v/v with an exposure time of 60 min (7). This tolerance to use concentrations of hydrogen peroxide has been attributed to the presence of several enzymes such as catalase (7). H. pylori is an uncommon bacterium, however, because it is only found in the human stomach.

On the other hand, Bacillus subtilis mutants (MBC = 0.5%) are more resistant to hydrogen peroxide than wild strains are (MBC = 0.34%) (8). This example shows that "resistant" means having reduced susceptibility. Mutant B. subtilis is still killed by hydrogen peroxide at use concentrations.

A strain of Pseudomonas aeruginosa survives exposure to hydrogen peroxide for as long as 60 min at a concentration of ~1.7% (9). But < 0.1% of the initial bacterial concentration survived. Use concentrations of hydrogen peroxide still kill the bacteria strain within minutes. The reduced susceptibility of P. aeruginosa to hydrogen peroxide is caused by its antioxidant defenses.

Hypochlorous acid. The presence of a biofilm (e.g., in potable-water pipes and cooling towers) significantly reduces the efficacy of hypochlorites. For example, LeChevallier reported that a bacterial biofilm grown on various surfaces were 150 to 3000 times more resistant to hypochlorous acid than planktonic cells were (10). Some types of bacteria such as Pseudomonads segregate extracellular mucopolysaccharides that enhance adhesion to solid surfaces. This is the hypothesized mechanism by which certain strains of P. aeruginosa resist disinfection with chlorine in swimming pools.

The hypothetical explanation for the reduced susceptibility and tolerance of bacteria within biofilms has two components: retarded penetration of chlorine into the biofilm and the consumption of free chlorine by organic materials before it can fully penetrate the biofilm surface (11). A similar theory explains the reduced susceptibility of biofilms to hydrogen peroxide.

Iodine. In an ostensible case of resistance to a disinfectant, the Centers for Disease Control (CDC) reported several peritoneal infections in infants and false-positive blood cultures from patients in 1989. These infections were associated with an iodophor antiseptic solution, namely povidone-iodine (PI), contaminated with Burkholderia cepacia (12). PI is a stable chemical complex of polyvinylpyrrolidone and iodine. A follow-up study of the PI solution confirmed the contamination and recovered the bacterium from the contaminated iodophor after 29 weeks of sampling. Scanning electron microscopic (SEM) examination of the contaminated PI solution showed bacterial cells embedded in extracellular material and strands of glycocalyx between cells.

The extended survival of B. cepacia in the PI solution was attributed to the extracellular glycocalyx-like material that microorganisms form and deposit on various surfaces (12). In this case, susceptibility and resistance to iodine were not reduced; bacteria were protected by extracellular materials. No report of true microbial tolerance or resistance to PI has been confirmed.

Alkaline solutions. Alkalis such as sodium hydroxide and potassium hydroxide kill bacteria through the action of hydroxide free radicals. In solution, sodium hydroxide and potassium hydroxide disassociate into metal cations and hydroxide free radicals, which oxidize lipids, proteins, and DNA.

Nonoxidizing disinfectants

Isopropyl alcohol (IPA) and ethyl alcohol (EA). Reduced susceptibility to IPA and EA at use concentrations in sensitive microorganisms has not been substantiated. Increase of the MIC—below use concentrations—has been documented for some gram-negative bacteria such as Escherichia coli and Salmonella species because of the oxidative-stress (SOS) response. The SOS response induces production of neutralizing enzymes to prevent cellular damage and to repair DNA injuries (13). Most alcohol disinfection failures in the field result from the presence of biofilms, poor cleaning, or inadequate contact times. IPA and EA are not effective in penetrating organic material and tissue. Hence, biofilms have reduced susceptibility, and sometimes resistance, to alcohols. The most common reason for efficacy loss with IPA and EA is evaporation that yields suboptimal contact times.

Formaldehyde (FA). Reduced susceptibility and resistance to FA are most often found in gram-negative bacteria such as Pseudomonas spp. and Enterobacteriaceae. A strain of P. aeruginosa grows at an FA concentration of 0.075% (14). A strain of E. coli grows at an FA concentration of 0.02% (15). Use concentrations of FA are 1–8% for immersion and wetting disinfection, and 0.05–0.2% for preservation. Studies of E. coli (strain VU3695) indicate that reduced susceptibility to FA is caused by enzymes such as FA dehydrogenases that inactivate it (15). Use concentrations of 1–8%, however, will readily kill these two bacterial strains.

The regrowth observed in vitro for FA donors, used in preservation, has several causes. First, FA kills most bacteria in the culture medium. Second, the available FA is exhausted after reacting with the organic molecules in the medium and the bacteria. Third, bacteria that survived because they were protected by organic material (i.e., cellular debris) and received a sublethal dose may acclimatize to residual FA and grow back. Bacteria that have FA dehydrogenases will degrade FA and keep multiplying. As the FA is exhausted from the medium, other bacteria will grow (14, 16). FA donors are mainly used as preservatives at concentrations of 0.05–0.20% (free FA).

Phenol. Some bacteria can survive in low concentrations of phenol. Furthermore, some of them can use phenol as a source of carbon and thrive in low phenol concentrations (< 0.3%). For instance, a strain of Micrococcus pyogenes var. aureus is resistant to the bacteriostatic action of phenol in 0.2–0.3% concentrations (17). In addition, a strain of Brevundimonas putida metabolizes phenol and the isomers of cresol at low concentrations (18, 19). At use concentrations, none of these bacteria would survive. In fact, tolerance to phenol has only been demonstrated at concentrations < 0.5% (20). True resistance or tolerance to phenol at use concentrations has not been documented.

Triclosan (TLN). TLN is a derivative of halogenated phenolics intended to be used as an antiseptic. It has poor solubility in water, but is fat-soluble and easily crosses cell membranes. Once inside microorganisms, TLN poisons a specific enzyme that many bacteria and fungi need to live. TLN blocks the active site of the enzyme enoyl-acyl carrier-protein reductase (ENR), preventing microorganisms from synthesizing fatty acids they need for building cell membranes and other essential functions (21, 22).

TLN's mechanism of action is considered the same as that of antibiotics because of the highly specific way that TLN kills microorganisms. TLN is thus different from common disinfectants, which do not have specialized cell targets. Clinicians and researchers are therefore worried about TLN's possible role in creating antibiotic-resistant strains of bacteria. Moreover, researchers have demonstrated that mutations in the bacterial gene that produces ENR can yield TLN-resistant bacteria (23–25).

Generally, TLN has little activity against P. aeruginosa, other gram-negative bacteria, and molds. Strains of Klebsiella pneumoniae,Enterobacter cloacae,Acinetobacter baumannii,Pseudomonas fluorescents, and E. coli–O157:H7 have grown on media containing TLN at a concentration of approximately 0.1% (24, 26). This concentration is near that of several consumer products. For example, some bars of soap contain 0.25–1.5% TLN by weight (27, 28).

Reduced susceptibility to TLN is seen in strains of Staphylococcusaureus. The typical TLN MIC for S. aureus is 0.016 μg/mL, but mutant strains had a MIC of 2 μg/mL: an increase of greater than fiftyfold. Payne revealed that a gene mutation is required for TLN resistance and that this gene must be overexpressed at levels three to five times higher than the level of expression in TLN-sensitive strains (23).

Although wild types of P. aeruginosa have the ENR enzyme, they are intrinsically resistant to TLN. Resistance comes from several factors, including efflux pumps and gene mutations (29, 30). Moreover, clinical and laboratory E. coli strains with a multidrug efflux pump have reduced susceptibility to TLN (24, 25). Efflux pumps are conveyor systems that move substances such as waste and harmful chemicals out of the cell.

Chlorhexidine (CHX). CHX compounds are low- to intermediate-level disinfectants and antiseptics. CHX interacts with the cell surface and promotes membrane damage, which in turn causes an irreversible loss of cytoplasmic components (31, 32). The killing action of CHX at relatively low concentrations (e.g., 2–2.5 μg/mL) is similar to the action of some antibiotics. At high concentrations (≥ 20 μg/mL), CHX causes coagulation of cytoplasm and precipitation of proteins and nucleic acids.

CHX at concentrations ≥ 20 μg/mL kills bacteria and yeasts (33). Reduced susceptibility to CHX among gram-positive bacteria is somewhat uncommon but has been reported in S. aureus (34). Conversely, gram-negative bacteria such as E. coli, Salmonella enteritica, Proteus mirabilis, Providencia stuartii, P. aeruginosa, B. cepacia, and S. marcescens (24, 35–39) have frequently shown reduced susceptibility to CHX and, in some cases, resistance at use concentrations. MICs as high as 1600 μg/mL have been reported, especially with strains of Providencia species (40).

Several gram-negative bacteria can be acclimatized to grow at low concentrations of CHX. They sometimes can thrive at or near use concentrations. One example is P. aeruginosa, which was exposed to 5 μg/mL of CHX. As a consequence of the exposure, the bacteria's MIC increased within six days from < 10 to 70 μg/mL (37). A second example is P. stutzeri, which reached a MIC of 50 μg/mL after 12 days of exposure to CHX (37). Wild strains of P. aeruginosa, K. pneumoniae, and A. baumannii grew at 1% CHX, which is within the recommended use concentration as an antibacterial agent.

Benzalkonium chloride (BKC). At low concentration in gram-positive bacteria (e.g., Listeria monocitogenes), BKC works by disrupting the membrane potential and pH gradient across the cellular membrane. At high concentration, BKC completely inhibits acidification and respiration and depletes adenosine triphosphate pools (41). In its mode of action, BKC resembles an antibiotic more than a disinfectant.

Several bacteria have exhibited reduced susceptibility to BKC, either because of intrinsic tolerance or by adaptation. Research indicates that efflux pumps are the major mechanism of adaptation of gram-positive bacteria such as S. aureus, L. monocitogenes, and other species of staphylococci. A strain of P. aeruginosa with resistance to BKC was shown to have defective porins. Hydrophilic molecules of low molecular weight usually can enter microorganisms by means of porins. P. aeruginosa may possess porins that do not function as do those in other bacteria.

A strain of B. cepacia showed a remarkable tolerance to BKC. This strain was isolated from a solution used as a cleansing or germicidal agent for catheters preserved with 0.05% BKC. The B. cepacia strain survived for 14 years in this solution. The strain's tolerance increased in increments of 0.5% to 16% BKC. The bacteria used BKC as a substrate for growth (42).

Development of resistance to disinfectants

True resistance or tolerance to common disinfectants such as chlorine, hydrogen peroxide, iodine, IPA and EA, phenol, and FA has not been documented. Logically, this must be the case because these disinfeectants attack microorganisms in various ways. True resistance or tolerance to antibiotic-like disinfectants (e.g., BKC, CHX, and TLN), however, has been documented empirically and experimentally (23–26, 29–30, 35–39, 42). These disinfectants, along with similar chemical agents, more closely resemble antibiotics than they do common disinfectants. On balance, their modes of action are basically the same as those of antibiotics. Bacteria can therefore develop resistance or tolerance to these agents.

One important mechanism of intrinsic reduced susceptibility and resistance or tolerance to antibiotic-like disinfectants and antibiotics in gram-negative bacteria and mycobacteria is the impermeability of the outer membrane or cell wall to these substances. In addition, many research studies have revealed that efflux pumps, at times with uncommonly wide specificity, contribute to the intrinsic resistance of gram-negative bacteria by removing agents such as detergents, antibiotics, and dyes from the cell (24, 25, 29, 30). An additional mechanism entails using enzymes to degrade harmful substances such as disinfectants and antibiotics. These enzymes can be intra- or extracytoplasmic. Another mechanism is the formation of biofilms. The other known mechanisms, which only apply to antibiotic-like disinfectants and antibiotics, are the modification of the target sites and substitution of susceptible metabolic pathways.

A microorganism can use any of the above mechanisms or a combination of them. These mechanisms are not new to microorganisms and do not result from the use of disinfectants and antibiotics. Evolution provides microorganisms with the necessary tools to survive in hostile environments. Microorganisms have struggled for food and survival for billion of years. Humans have unwittingly selected the fittest microorganisms through the overuse and abuse of antibiotics and antibiotic-like disinfectants.

Cross-resistance (i.e., tolerance to a toxic substance as a result of exposure to a similarly acting substance) has been proven only for the antibiotic-like disinfectants. Mutant strains of S. aureus that were resistant to BKC showed a higher resistance than their parent strains to various ß-lactam antibiotics and to ofloxacin, a quinolone (43). One strain of Salmonella typhimurium that developed resistance to CHX also became resistant to erythromycin and BKC (24). In the same study, another strain of Salmonella developed a high degree of cross-resistance to other antibiotics and biocides.

Adaptive cross-resistance to BKC, amikacin, and tobramycin also has been documented (44). Exposure of a P. aeruginosa strain susceptible to TLN resulted in a multidrug-resistant bacteria at high frequencies (45). Because of gene mutations, the strain hyperexpressed an efflux-pump system. The MICs of tetracycline, trimethoprim, erythromycin, and gentamicin for the mutants were increased by as much as 500 times. The MIC of ciprofloxacin, a quinolone, was increased 94 times.

Antibiotics

In contrast to disinfectants, antibiotics are substances that are selectively toxic for bacteria. Bactericidal antibiotics kill them, and bacteriostatic antibiotics inhibit their growth without harm to the patient. These substances must act on targets found in bacteria, not in the patient. This characteristic distinguishes antibiotics from disinfectants. Antibiotics work best in conjunction with a healthy immune system to kill infecting bacteria in the host (46).

For antibiotics to be effective, the MIC or MBC must be reached at the site of infection. The pharmacological properties of the antibiotic influence the route and dose to achieve a successful concentration at the site of infection. Antibiotics are divided into several classes, each with its own mode of action. This article only discusses the major classes of antibiotics that are active against bacteria because they provide a good example for comparison with disinfectants.

Antibiotics that bind to the 30S ribosomal subunit. Aminoglycosides. The most common aminoglycosides are streptomycin, kanamycin, gentamicin, tobramycin, amikacin, netilmicin, and neomycin, which are bactericidal. They are active against many gram-negative and some gram-positive bacteria, but resistance to them is common.

Their mode of action is twofold. The initial site of action is the outer membrane (OM). Antibiotic molecules create crevices in the OM, resulting in leakage of intracellular contents and enhanced antibiotic uptake (47, 48). Their quick damage to the OM probably contributes to aminoglycosides' bactericidal activity. Second, aminoglycosides irreversibly bind to the 30S ribosomal subunit and freeze the 30S initiation complex (30S–mRNA–tRNA) so that no further initiation can occur (49).

Aminoglycoside resistance results from production of aminoglycoside-modifying enzymes, reduced uptake or decreased cell permeability, and alterations at the ribosomal binding sites. Most resistance to aminoglycosides is caused by bacterial inactivation by intracellular enzymes (50, 51).

Tetracyclines. Tetracycline and doxycycline, the most common examples, are bacteriostatic. Tetracycline's mode of action is to reversibly bind to the 30S ribosome and inhibit the binding of aminoacyl-t-RNA to the 70S ribosome. Tetracyclines' spectrum of activity is broad, but resistance is common. Resistance occurs through ribosomal protection and efflux pumps.

Ribosomal protection results from minor changes in the ribosome that prevent the drug from binding to it and stop the production of new proteins. The bacteria can thus continue as if the drug were not there (52, 53). The second form of resistance, efflux pumps, is especially common in gram-negative bacteria. As the drug enters the cell through its porins, efflux pumps pump it out of the cell (54, 55). Again, the bacterium behaves as if the drug was not present. The net result is that the drug does not inhibit protein production. Cross-resistance, resulting from these two mechanisms, to all of the drugs in the tetracycline class is widespread.

Antibiotics that bind to the 50S ribosomal subunit. Chloramphenicol subclass. This subclass includes chloramphenicol (CAM), lincomycin, and clindamycin, which are bacteriostatic. CAM is active against gram-positive and gram-negative bacteria, but lincomycin and clindamycin have a restricted spectrum of activity. Resistance to all three is common.

CAM's effect on bacteria stems from the inhibition of bacterial protein synthesis (56). The molecular target for CAM is the peptidyl transferase (PTF) enzyme that links amino acids in the growing peptide chain (57). The antibiotic halts the process of chain elongation, thus stopping bacterial growth (58, 59). The process is completely reversible, and CAM is fundamentally a bacteriostatic agent. The binding of CAM to the 50S subunit of 70S ribosomes is highly specific.

Many bacteria are resistant to CAM. The main mechanisms for resistance are covalent modification of the antibiotic by the enzyme chloramphenicol acetyl transferase or CAT (so that the antibiotic no longer binds to ribosomes) and efflux pumps (60).

Macrolides. Macrolides bind specifically to the PTF site. This group includes erythromycin (ERY), azithromycin, clarithromycin, and dirithromycin. ERY is the best known agent of this group. It is a bacteriostatic agent that is only active against gram-positive bacteria, Mycoplasma spp. and Legionella spp. Resistance is common. Depending on drug concentration, bacterial species, and phase of growth, ERY may act in different ways. The main modes of action for ERY, and macrolides in general, are the following:

• Large macrolides bind near the PTF center and block the nascent peptide from emerging from the ribosome, thus inhibiting the elongation of most peptides and destabilizing the ribosome–peptidyl–tRNA complex.
• Small macrolides partially block the ribosome channel and inhibit peptide elongation depending on the conformation of the nascent peptide.
• The nascent peptide chain becomes stuck inside the ribosome, thus inhibiting protein synthesis.
• The ribosome-peptidyl-tRNA complex that inactivates the ribosome is stabilized (61).

Bacteria are resistant to macrolides in at least three ways. Methylation can modify the target site and prevent the antibiotic from binding to its ribosomal target. Efflux pumps and drug inactivation also provide resistance (62, 63).

Antibiotics that interfere with elongation factors. Fusidic acid (FAc) is a steroid-like bacteriostatic antibiotic (64). It inhibits the translocation step in protein synthesis by binding to elongation factor G or EF-G (65, 66). Resistance results from the following two causes:

• Production of an altered EF-G with decreased FAc affinity (67)

• Extracellular enzymes that inactivate FAc (68).

Inhibitors of nucleic-acid synthesis and function. Inhibitors of RNA synthesis and function. These antimicrobials bind to DNA-dependent RNA polymerase (DDRP) and inhibit the initiation of RNA synthesis. They have a wide spectrum of activity, but are most commonly used to treat tuberculosis. These agents are bactericidal, but resistance is common. This group includes rifampin, rifampicin, rifabutin, and rifapentine.

The most popular agent in this group is rifampin, which binds to the RNA polymerase and inhibits transcription (69). Mutations in the DDRP gene are associated with rifampin resistance (70). Isolates resistant to rifampin have reduce susceptibilities to rifapentine, rifabutin, and other rifamycins (71). Hence, cross-resistance among the rifamycins is common.

Inhibitors of DNA synthesis and function. Quinolones are bactericidal. The first quinolone, nalidixic acid, was introduced in 1962 (72). Since then, modifications to the structure of nalidixic acid have resulted in second-, third-, and fourth-generation fluoroquinolones that have improved activity against gram-positive, gram-negative, and anaerobic bacteria (73–75).

Target sites of quinolones are DNA gyrase (topoisomerase II) and topoisomerase IV or Top IV (76–81). DNA topoisomerases are enzymes that control and modify the topological states of DNA in cells. DNA gyrase catalyzes the negative supercoiling of prokaryotic DNA (82, 83). This supercoiling is essential for initiating and continuing DNA synthesis and for the transcription of mRNA from genes. Top IV, unlike DNA gyrase, cannot catalyze the supercoiling of DNA; it can only catalyze its relaxation. Top IV is responsible for cell division after DNA replication (84, 85).

Quinolones form a complex with DNA and DNA gyrase or Top IV (84). Quinolones inhibit the release of DNA from DNA gyrase, prevent the rebinding of DNA strands, and break DNA (84, 85). As a rule of thumb, gram-negative bacterial activity correlates with the inhibition of DNA gyrase, and gram-positive bacterial activity corresponds with the inhibition of Top IV.

Resistance to quinolones results from mutations in the genes' encoding for DNA gyrase and Top IV. Gene mutations change the primary structure of DNA gyrase and Top IV. These small changes prevent the enzymes from binding to quinolones and forming a quinolone–DNA–DNA gyrase (or Top IV) complex.

Inhibitors of bacterial-wall synthesis (β-lactam). The β-lactam antibiotics are natural and semisynthetic compounds that inhibit several enzymes associated with the final step of peptidoglycan synthesis and assembly. These antibiotics are derived from the β-lactam structure: a four-tiered ring in which the β-lactam bond resembles a peptide bond. The β-lactam group includes the following five subgroups:

• Penicillins (e.g., 6-aminopenicillanic acid derivatives)

• Cephalosporins (e.g., 7-aminocephalosporanic acid derivatives)

• Cephalosporin-related agents (e.g., oxacephems and cephamycins)

• Carbapenems (e.g., imipenem)

• Monobactams (e.g., aztreonam) (86).

The β-lactam antibiotics inhibit bacterial-wall synthesis. The targets for β-lactam antibiotics are the penicillin-binding proteins (PBPs), which are permanently inactivated. The PBPs have transpeptidase or carboxypeptidase activity that regulates cell size and shape. In addition, PBPs are involved in cell division through the manufacture of new bacterial walls. Bacteria have many PBPs, and each one has a separate function.

The following are the four major mechanisms of resistance to β-lactam antibiotics:

• Inactivation by β-lactamases

• Modification of target PBPs

• Efflux pumps

• Reduced OM permeability (86, 87).

Antimetabolites: inhibitors of folic-acid synthesis. The specificity of these antibiotics stems from the fact that bacteria cannot use preformed folic acid and must synthesize their own folic acid (88). Mammalian cells use folic acid from exogenous sources.

Sulfonamides. These antibiotics are analogues of paraaminobenzoic acid (PABA). Sulfonamides block bacterial reproduction by acting as competitive inhibitors of PABA in the folic-acid metabolism cycle. The target molecule is dihydropteroate synthase (DHPS). Inhibition of this enzyme results in the depletion of tetrahydrofolate (89). Therefore, sulfonamides are bacteriostatic. Sulfonamides possess a broad range of activity against gram-positive and gram-negative bacteria. Sulfamethoxazole is the most popular drug in this subgroup of antibiotics.

Resistance is common, and resistance to one sulfonamide provides resistance to all. Sulfamethoxazole resistance is the result of either chromosomal mutations in the DHPS gene that yield drug-resistant forms of DHPS or efflux pumps (90–92).

Sulfanilamides. These antibiotics bind to dihydrofolate reductase (DHFR) and inhibit the formation of tetrahydrofolic acid. They are thus bacteriostatic. Their spectrum of activity covers gram-positive and gram-negative bacteria. Trimethoprim (TMP) is representative of this subgroup.

The major causes of TMP resistance are the following:

• Production of an altered DHFR that lacks the capacity to bind sulfanilamides

• Decrease in drug uptake

• Efflux pumps

• Overproduction of the normal DHFR (93–95).

Development of resistance to antibiotics

Resistance to antibiotics develops in only two major ways. The antibiotic, acting as a selective agent, can help propagate resistance microorganisms. In other cases, particular genes provide resistance. If either of these two conditions is not present, resistant bacteria are not developed.

Bacterial resistance to antibiotics is a natural outcome of selective pressure or evolution. Bacterialike organisms arose more than three billion years ago and have been constantly faced with natural products that have antibacterial activity (i.e., antibiotics) and related organic molecules. Bacteria have acquired resistance genes to survive. When a random mutation provides a bacterium with a resistance gene, its progeny will have a survival advantage and proliferate despite the presence of substances that inhibit or kill competing microorganisms. Resistance genes are passed to other bacteria through various gene-transfer processes. In general, bacterial resistance to antibiotics is either intrinsic (i.e., resulting from native genes) or acquired (i.e., resulting from new genes).

Conclusion

Reduced susceptibility to a disinfectant does not mean that the agent or the disinfection method fails. Most of the evidence for resistance to disinfectants, antiseptics, and sanitizers is laboratory-based. Little, if any, evidence has been gleaned from real-life situations. Resistance in laboratory situations means reduced susceptibility.

Common disinfectants, antiseptics, and sanitizers are used at high concentrations in real-life to attain swift microbicidal action and produce effects such as disruption of the cellular membrane or wall, inactivation of critical enzymes, and degradation of DNA or RNA. At lower concentrations, these substances inhibit microorganisms' growth.

Rotation of disinfectants in the pharmaceutical and biotech industries has been promoted to prevent the development of bacterial resistance. The argument is that one disinfectant should be replaced by another that has a different mode of action. This recommendation is derived from experience with antibiotics that does not apply directly to disinfectants, antiseptics, and sanitizers. The reported resistance to common disinfectants does not occur at use concentrations, and is more accurately considered reduced susceptibility (96, 97).

This assessment agrees with a report issued by CDC on Oct. 28, 1997, which said, "Antibiotic resistant microorganisms are susceptible (or killed) to chemical germicides. The mechanisms by which chemical germicides and antibiotics work are completely different and there does not seem to be a relationship between antibiotic resistance and chemical germicide effectiveness" (98).

Because common disinfectants are used on lifeless objects rather than on living tissue, they are used at concentrations that exceed the MIC or MBC by several orders of magnitude. Consequently, a decrease in susceptibility by a factor of two or more, which is important to an antibiotic, has no relevance to the effectiveness of a common disinfectant.

Rotation of a common disinfectant and a sporicidal helps ensure that bacterial spores do not take hold in manufacturing and aseptic areas. But the rotation of common disinfectants such as those based on phenol-derivatives (except TLN), aldehydes, and oxidizing agents, has no scientific basis. If antibiotic-like disinfectants are used, however, rotation is a necessity.

The development of resistance to antibiotics has been extrapolated to common disinfectants, antiseptics and sanitizers, and the general environment. A misunderstanding of the vocabulary related to disinfectant and antibiotic susceptibility tests seems to be the justification for this extrapolation. The elemental differences between disinfectants' and antibiotics' mechanisms of action and the methods used to evaluate their efficacy are often left unconsidered. The lack of standard terminology for interpreting studies can result in inaccurate interpretations of the data.

José E. Martínez is a consultant at JEM Consulting Services, Box 4956 PMB 652, Caguas, PR 00726, tel. 787.349.3857, martinez_jose_e100@yahoo.com

Submitted: Apr. 29, 2008. Accepted: Aug. 3, 2008.

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References

1. P. Gilbert and A.J. McBain, "Potential Impact of Increased Use of Biocides in Consumer Products on Prevalence of Antibiotic Resistance," Clin. Microbiol. Rev. 16 (2), 189–208 (2003).

2. "Annex 1: Manufacture of Sterile Medicinal Products," Good Manufacturing Practice (GMP) Guidelines (Brussels, May, 2003),

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