Antimicrobial Disinfectant Formulations

Disinfectants are a class of antimicrobial chemicals which are used to kill microorganisms inhabiting non-living objects and surfaces; they are distinct from antibiotics and antiseptics which are employed to kill microorganisms living inside the body or on living tissues. Disinfectants are widely used in industry, hospitals and domestically to ensure hygiene in the home setting. An ideal disinfectant would be one which sterilises (eliminate 100% of microorganisms) with minimal toxicity to humans and animals and minimal damage to the surface being cleansed. In reality, such a disinfectant does not exist and a balance is struck between effectiveness and practicality of use. The choice of disinfectant employed generally depends on the degree of sterility required and the characteristics of the surface to be disinfected.
Disinfectants are available in many forms including physical factors (such as x-rays, ultraviolet radiation or heat) and chemical compounds (including alcohols, aldehydes, halogens, oxidising agents, phenolics and quaternary ammonium compounds) (McDonnell and Russell, 1999). Phenol is one of the oldest known disinfectants and effective against most bacteria and enveloped viruses. While phenol based disinfectants are generally safe, prolonged skin exposure may cause irritation. Alcohols are less irritating and are effective bactericidals employed as surface disinfectants in hospitals and laboratories; however they do carry an ignition risk. Halogens such as hypochlorites are amongst the most common disinfectants and are a cheap and effective solution for many situations. Chlorine based bleaches have a wide spectrum of antimicrobial activity. While toxicity is low, chlorine gas can be released during use and hypochlorites are corrosive to many metals. Aldehydes are highly effective disinfectants however they can also be highly toxic and require special care in their application. Gamage (2003) reviewed the different available antimicrobial formulations.
Disinfectants work through a diverse selection of biochemical mechanisms. Some work at the surface of the microbial cell such as glutaraldehyde which interacts with the outer layers of organisms such as bacteria and works through the crosslinking of exposed proteins, inhibiting a variety of essential cell functions. Disruption of the cellular membrane (using phenol or quaternary ammonium compounds) is an effective method for lysing cells. Many disinfectants work intracellularly by interfering with vital cellular processes such as DNA synthesis (halogens), crosslinking of cellular proteins (formaldehyde) or through general intercellular damage through oxidation (halogens) or the generation of reactive free radicals (peroxygens) (McDonnell and Russell, 1999).
The effectiveness of disinfection depends on three factors: the chosen disinfectant, the characteristics of the target microorganisms, and the method of application. Individual disinfectants may be effective against most known microorganisms or targeted against a specific class of microorganism. In general, higher concentrations of product will be more efficacious; however these may result in toxicity or damage to the surface. The effectiveness of all disinfectants depends on direct contact with the target microbes, so factors relating to the application method such as contact time and degree of contaminating dirt can all influence how much disinfectant is applied to the microorganisms. Additionally, general chemical factors such as temperature, humidity, pH and the age and storage conditions of the disinfectant will alter its effectiveness.

Quaternary Ammonium Compounds

Quaternary ammonium compounds (QACs) are salts formed through the reaction of a ‘quaternary ammonium cation’ with an anion. A quaternary ammonium cation is a compound consisting of four alkyl groups bonded to a central nitrogen atom. Nitrogen normally forms 3 covalent bonds and thus the presence of four bonded alkyl groups gives the nitrogen atom a positive charge. This gives the QAC the unique property of being simultaneously both polar (due to the charged nitrogen ion) and non-polar (due to the non-polar alkyl chains). In chemistry, quaternary ammonium compounds are the most commonly used ‘phase transfer catalysts’, which facilitate reactions between normally immiscible compounds.
The polar/non-polar property of QACs bestows powerful antimicrobial properties through the ability to disrupt the microbial phospholipid bilayer. The positively charged nitrogen ion associates with the polar head groups of the membrane phospholipid molecules whilst the non-polar alkyl groups associate with the hydrophobic tails (McBain et al, 2004). This causes perturbation of the phospholipids in the membrane resulting destruction of the lipid bilayer and cellular lysis (Przestalski et al, 2000). QACs are widely employed as antimicrobials and have broad-spectrum antimicrobial activity. Quaternary Ammonium Compounds are effective at low concentrations and their low toxicity, lack of odour and associated detergent effect renders them a good choice for a general cleaning and disinfecting agent; however they are of limited effect against non-enveloped viruses and fungi.

Ethylenediaminetetraacetic

Ethylenediaminetetraacetic acid (EDTA) is widely used as a chelating agent in industry and medicine. The chemical structure of EDTA allows it to ‘wrap around’ (chelate) a variety of large metal ions effectively sequestering them and rendering them water soluble. This property bestows EDTA with a variety of applications including as a treatment for lead or mercury poisoning, as a water softener (through the removal of Ca2+ and Mg2+ ions) and as a preservative to prevent oxidation by catalytic metal ions.
EDTA is also an effective disinfectant and can induce permeabilization of the outer membrane of gram-negative bacteria resulting in cell lysis. The exact mechanism by which EDTA lysis cells is not clear, however it is believed that chelation of metal ions from the outer membrane results in dissociation of lipopolysaccharides and protein components from the cell membrane (Vaara, 1999). Prachayasittikul (2007) proposed an additional mechanism for cellular lysis based on the interaction of EDTA with the polar head groups of membrane lipid molecules separating the closely packed phospholipids resulting in ‘fluidization’ of the lipid bilayer and subsequent breakdown of the bilayer structure. This property of EDTA may explain the synergistic effect seen when EDTA is given simultaneously with antibiotics, increasing membrane permeability would be expected to dramatically increase the efficacy of antibacterials.

Minimum Inhibitory Concentration

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial agent which effectively inhibits the visible growth of a microorganism following overnight incubation. MICs are determined using a serial dilution of the antimicrobial added to a standardized culture of the relevant microbe (Andrews, 2001). Culturing can be performed using either solid agar plate cultures or a liquid broth culture, however, in order to overcome the complexities involved in producing multiple dilutions of nutrient containing media, many laboratories now employ the ‘agar diffusion test’. This simple test uses a single agar dish inoculated with the test strain onto which a disc of filter paper impregnated with the antimicrobial agent is placed. The agent diffuses through the agar medium (reducing in concentration as it diffuses away from the disc) and the MIC can be calculated through measurement of the diameter of the region of microbial growth inhibition (Kayser, 2005).
MICs are used to determine an in-vitro measurement of the effectiveness of newly developed antimicrobials; however, they can also be used as a guide to establishing an effective working concentration for disinfection, or suitable dose for the clinical application of antibiotics. Public health laboratories also monitor MICs of known antimicrobial agents to identify the development of resistance to classical antimicrobial agents.

Bacterial Resistance to Antimicrobial Disinfectants

Bacterial resistance to antimicrobials can broadly be split into intrinsic resistance and acquired resistance. Intrinsic resistance mechanisms reflect the different biological characteristics of different species of bacteria such as the presence of a second, outer membrane in gram-negative bacteria which acts as a barrier to the entry of a variety of antibacterial agents rendering them intrinsically more resistant than gram-positive bacteria. Other intrinsic characteristics of bacteria which increase their ability to thrive in the presence of antimicrobials include the ability to sporulate and the ability to exist in the protective environment of a biofilm.
Acquired resistance is a form of evolution and is achieved either through mutation of the bacteria’s own genome or through the acquisition of additional genes in the form of plasmids or transposons (Russell, 1999). This results in changes in gene expression within the microbe such as the expression of inactivating enzymes which allow the resistant microorganism to hydrolyse or modify the antimicrobial agent. Expression of ß-lactamases can hydrolyse the ß-lactam ring of antibiotics such as penicillin and is a common mechanism by which many bacteria gain resistance to penicillin (Koch, 2000). Resistance to formaldehyde based disinfectants has been demonstrated to be due to both alterations in the cell surface of the bacterium and expression of formaldehyde dehydrogenase (McDonnell and Russel, 1999). Increased removal of the antimicrobial from the cell cytoplasm through upregulation of drug efflux pumps is an alternative mechanism for generating resistance to a variety of disinfectants and antibiotics (Piddock, 2006).

Biofilms

A biofilm is a closely packed aggregate of microorganisms growing as a complex attached to each other and usually some solid surface. Biofilms are important in both industry (such as the soiling of water pipes due to biofilm build up) and in clinical situations. Most in-vivo populations of bacteria grow as an adherent biofilm, as opposed to in-vitro cultures of microorganisms which generally exists as free floating single cells in a liquid medium (Reisner et al, 2005). Formation of a biofilm begins with the attachment of single-celled free floating microorganisms, followed by modification of the surface generating more diverse adhesion sites which promotes rapid colonisation by other microbes and progeny of already attached microorganisms (Amano et al, 1999).
Biofilms are characterised by the presence of an extracellular matrix, produced through the excretion of polymeric compounds by constituents of the biofilm, and altered physiological and biochemical properties between the free floating and attached forms of individual microorganisms (Donlan and Costerton, 2002). One of the most clinically important altered properties of microbes living in biofilms is increased resistance to common antimicrobials (Stewart and Costerton, 2001). The mechanisms by which this resistance arises are still unclear; however it is likely that the dense extracellular matrix provides protection to microbes embedded in the biofilm through reduced penetration of antimicrobials (del Pozo and Patel, 2007). Limited availability of nutrients to microorganisms embedded in the biofilm forces a reduction in the growth rate of those microbes which results in physiological and phenotypic variation of the microbes and influences their susceptibility to common antimicrobials.
Biofilms are involved in a wide variety of microbial infections with data from the NIH suggesting that ‘more than 60% of all microbial infections are caused by biofilms’ (Costerton et al, 1999). These included common urinary tract infection, middle ear infection, gingivitis and common dental plaque. The microorganism constituents of biofilms are able to colonise virtually any surface material and this poses significant infection risks for urinary catheters, mechanical heart valves, contact lenses and other medical devices (Donlan, 2001). Biofilms are difficult to eradicate and the source of many refractory infections.

Staphylococcus Aureus

Staphylococcus aureus is a spherical, gram-positive coccus, bacteria which grows as clusters of cells. Asymptomatic colonization of humans is common with 20-25% of the population carrying this bacterium on the skin or the lining of the nasal cavity (von Eiff et al, 2001). S aureus causes many minor complaints including skin infections, pimples, boils, as well as more serious conditions including pneumonia, meningitis, food poisoning, toxic shock syndrome and septicaemia. In addition S aureus is one of the most common causes of post surgical wound infection. Diagnosis of S aureus infection involves Gram staining followed by enzyme tests to identify catalase, coagulase, DNase, lipase and phosphatase, characteristic enzyme constituents of S aureus.
Antimicrobial resistance is a major characteristic of S aureus. Resistance to Penicillin is mediated by expression of the blaZ gene, which produces the penicillinase enzyme (Kernodle, 2000). Penicillinase-producing strains of S aureus were first identified in the mid 20th century and penicillin resistance has gradually increased since with 40% incidence in the 1940’s, 80% by the 1980’s, and today only 2% of strains are sensitive to penicillin (Chambers, 2001). Of particular clinical concern today is the emergence of MRSA (methicillin resistant S aureus), a major public health concern with mortality rates of between 20 and 40% (Crowcroft and Catchpole, 2002). Methicillin was introduced in 1961 and the evolution of resistance to this semi-synthetic penicillin derivative was rapid, with outbreaks of resistant strains being reported in the same year. Although methicillin is no longer used in the treatment of S aureus, MRSA remains a major health problem due to the resistance of this strain to multiple and diverse antibiotics. Novel treatments have been proposed to try to prevent nasal colonization of S aureus in asymptomatic carriers including the use of bacteriophages as an antibacterial therapeutic (Fischetti, 2001), and vaccination as a potential future prospect for preventing S aureus infection (Fattom et al, 2004).