Bacterial resistance to biocides has been reported since the 1950s, particularly with the contamination of cationic biocide formulations. In most instances bacterial resistance emerged following the improper use or storage of the formulations, resulting in a decrease in the effective concentration. Bacterial resistance to all known preservatives has also been reported.
In the health care setting, bacterial resistance to biocides has long been reported with compounds such as: chlorhexidine ; quaternary ammonium compounds; bisphenol, triclosan ; iodophor ; parabens ; and more reactive biocides such as glutaraldehyde and peroxygens . In a recent study, Smith and Hunter reported that although biocides may be effective against planktonic populations of bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, some biocides currently used in hospitals are ineffective against nosocomial pathogens growing as biofilms attached to surfaces and fail to control this reservoir for hospital-acquired infections. Concerning triclosan, Tabak and colleagues reported that the tolerance of Salmonella in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance both to triclosan and to other antimicrobials.
However, most of the evidence on bacterial resistance to biocides comes from laboratory-based experiments which investigated a wide range of agents such as: cationic biocides; isothiazolones ; phenolics; hydrogen peroxide and peracetic acid and other compounds.
3.4.2. Biocide concentration and bacterial susceptibility
The concentration of a biocide has been deemed to be the most important factor that affects its efficacy. In the case of bacterial biofilms, the biocide concentration and consequently the bacterial susceptibility, is strongly affected by the reduced diffusion of active molecules through the biofilm . Concentration is also central to the definition of bacterial resistance in practice. Therefore, the measurement of bacterial lethality rather than the measurement of bacterial growth inhibition is paramount.
Many reports on emerging bacterial resistance to biocides are based on the determination of minimum inhibitory concentrations (MICs). Using MICs to measure bacterial resistance is arguable since much higher concentrations of biocides are used in practice and, therefore, failing to achieve a reduction of bacterial numbers (i.e. lethality) because of elevated MICs is unlikely. Indeed, some studies have shown that bacterial strains showing a significant increase in MICs to some biocides were nevertheless susceptible to higher (in use) concentrations of the same biocides.
Thus, the determination of minimum bactericidal concentrations (MBCs) is a more appropriate methodology that allows the comparison of lethality between a standard and the resistant strains. Here the standard strains represent the population of bacteria which is normally susceptible to the biocide.
Likewise, the determination of the lethality of the in-use concentration of a biocide will indicate whether a bacterial strains is insusceptible (i.e. naturally resistant) or resistant (by comparison to a standard strain). The determination of the inactivation kinetic following exposure to a biocide, and in particular the shape of the inactivation curve, will provide information as to the nature of resistance of a population of cells and/or the interaction of the biocide with the cell population.
The determination of the lethality of a biocide must involve the use of a neutralising agent or the removal of the biocide. Failure to do so will provide an over-estimation of the lethality of the biocide.
MIC determinations have been used in many studies as an indicator of bacterial sensitivity change to a biocide.
Bacteria showing an increased low-level of resistance/tolerance to a biocide might be selected by a low concentration of a biocide. Their level of resistance can increase through selection, for example by repeated exposure to a low concentration of a biocide or to increasing concentrations of a biocide.
The determination of bacterial growth kinetics in the presence of a low concentration of a biocide can also provide indications of a change in bacterial phenotype.
Table 7 highlights the methodologies that have been used to measure bacterial resistance to biocides