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Aims: To investigate the inhibitory and lethal effects of triclosan against several micro-organisms at different stages of their phase of population growth., Methods and Results: Triclosan minimum inhibitory concentrations against several test organisms were determined in broth and agar using standard protocols. The bisphenol effect on bacterial population growth kinetics was studied using the Bioscreen C microbial growth analyser. Finally, the efficacy of triclosan on phases of bacterial growth was determined using a standard suspension test. The duration of the lag phase for all micro-organisms tested was increased by bisphenol in a concentration-dependent manner. The population growth kinetics of the micro-organisms was also altered after biocide exposure. At higher concentrations, triclosan was bactericidal regardless of their phase of population growth, although population in stationary phase and particularly, washed suspensions, were more resilient to the lethality of triclosan. This lethal activity was concentration and contact time dependent, and in some instances, bactericidal activity of bisphenol was observed within 15 s., Conclusions: Low concentrations of triclosan affected the growth of several bacteria, while higher concentrations were bactericidal regardless of the bacterial phase of population growth., Significance and Impact of the Study: Here, we presented clear evidence that the interaction of triclosan with the bacterial cell is complex and its lethality cannot be explained solely by the inhibition of metabolic pathways such as the enoyl acyl-reductase. However, the inhibition of such pathways cannot be ruled out as part of the lethal mechanism of the bisphenol at a low bactericidal concentration.

Ortho-phthalaldehyde (OPA) is a new aromatic dialdehyde antimicrobial agent, the mechanism of action of which has been little studied. The aims of this paper are to examine what is currently known about its mechanism of action, to compare the action with that of a widely investigated aliphatic dialdehyde, glutaraldehyde (GTA), and to put forward a hypothesis that would, in the light of current knowledge, explain how OPA inactivates micro-organisms, including GTA-resistant Mycobacterium chelonae.

Agents which had previously been shown to act as permeabilizers against Pseudomonas aeruginosa or other Gram-negative bacteria were tested to determine whether susceptibility to various antibiotics could be increased. In the absence of a permeabilizer, Ps. aeruginosa was resistant to several hydrophobic antibiotics and vancomycin, but not to gentamicin. Tartaric and gluconic acids had weak potentiating activity, whereas ethylenediamine tetraacetic acid and citric acid were more effective permeabilizers. However, sodium polyphosphate enhanced the activity of erythromycin, fucidin, novobiocin, rifampicin and methicillin; vancomycin was unaffected and the activity of gentamicin was reduced considerably.

The presence of divalent (Mg2+) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7.8 or 9.0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg2+ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.

A simple Pseudomonas aeruginosa G48 biofilm on stainless steel discs provided a useful primary screen of potentiating effects of various permeabilizing agents on antibacterial agents. Experiments with Ps. aeruginosa suspensions could not be used to predict the effects of biocides and permeabilizers on biofilms. Although antibacterial activity against biofilms was less than demonstrated in suspension tests, potentiation by some permeabilizers was still observed.

The Malthus-AT system provided a satisfactory method for examining the effects of permeabilizing agents on the activity of sub-inhibitory concentrations of antibacterial agents against Pseudomonas aeruginosa G48. Under this system, disodium edetate potentiated the activity of chlorhexidine diacetate (CHA), cetylpyridinium chloride, para-chlorometaxylenol and triclosan. Nitrilotriacetic acid enhanced the activity of some of the antibacterials tested, whereas sodium polyphosphate markedly reduced the efficacy of CHA.

Microbiologically it was demonstrated that amino acids, e.g. cysteine (CySH), and other compounds, e.g. sodium thioglycollate, containing thiol groups neutralized the activity of silver nitrate against Pseudomonas aeruginosa PAO1. Amino acids with disulphide bonds were inactive, with the exception of L-cystine dimethyl ester, as were all amino acids with no sulphur groups. Iodoacetamide reacted with CySH to produce a CyS-acetamide complex that was unable to quench the activity of Ag+. Chemical analyses using cyclic voltammetry demonstrated that high coordination numbers (3.1) were obtained with thiol-containing amino acids and low numbers (0.28-0.4) with other amino acids. Both microbiologically and chemically, the results imply that interaction of Ag+ with thiol groups plays an essential role in bacterial inactivation.

The effects of sublethal concentrations of the membrane-active agent chlorhexidine gluconate (CHG) on the growth rate and sporulation of Bacillus subtilis vegetative MB2 cells have been investigated. CHG increased the mean generation time (Mgt) of vegetative cells in casein medium. It also affected spore development: as CHG concentrations increased, spore index (SI) values decreased and sensitivity to both toluene and heat increased.

Uptake of radiolabelled chlorhexidine gluconate (14C-CHG) to Saccharomyces cerevisiae, Candida albicans and C. glabrata was very rapid and near maximal within 30 s. The organism, S. cerevisiae, most sensitive to the lethal action of chlorhexidine, took up significantly more biocide than the other organisms. Cells from cultures of different ages took up different amounts of 14C-CHG.

The use of energy dispersive analysis of X-rays (EDAX) to identify and quantify the presence of chlorhexidine diacetate (CHA) within Saccharomyces cerevisiae cells was examined. Chlorine was used as the elemental marker tag. Saccharomyces cerevisiae cells exposed to 1000 micrograms ml-1 CHA took up increasing amounts of CHA over a time period of 30 s to 30 min. Electron probe micro-analysis was employed to examine the specific accumulation of CHA across the treated cells. These results showed that CHA was distributed evenly between the cell wall, cytoplasm and vacuoles of cells pre-treated with this concentration of CHA for 30 min. The EDAX system therefore provides a useful tool for examining the qualitative and quantitative effects of chlorhexidine on yeast cells, although quantitative data must be interpreted with caution.

Using an energy dispersive analyser of X-rays fitted to a scanning electron microscope, chlorhexidine was shown not to bind onto F116 bacteriophage, unlike cetylpyridinium chloride, which possibly penetrated the phage. This could explain the difference in viricidal activity between the two compounds.