A. Denver Russell

Triclosan has activity against many, but not all, types of Gram-positive and Gram-negative bacteria. It is bacteriostatic at low concentrations, but higher concentrations are bactericidal. Pseudomonas aeruginosa is highly resistant, whereas methicillin-resistant Staphylococcus aureus strains are inhibited over a range of ∼0.1–2 mg/L. Triclosan shows significant activity against some mycobacteria, but is not sporicidal. Its growth-inhibitory properties result from an inhibition of enoyl reductase, FabI. Membrane-destabilizing effects are likely to be responsible for bacterial inactivation by higher concentrations. Resistance can arise from mutations in, and/or overproduction of, FabI, impermeability or efflux. Whilst triclosan resistance in laboratory experiments may be associated with changes in antibiotic susceptibility, comprehensive environmental surveys have not demonstrated any association between triclosan usage and antibiotic resistance. Triclosan has several important uses, and the future aim must be to retain these applications whilst eliminating the more frivolous and unnecessary ones.

Unlike antibiotics, biocides are multi-targeted antimicrobial agents. Several of the damaging effects reported to occur in the most widely studied organisms, bacteria, may also take place to varying degrees in other organisms. Nevertheless, there is considerable variation in the response of different microorganisms to biocides. Bacteria themselves (Gram-positive and Gram-negative vegetative organisms, mycobacteria and spores) respond differently to biocides and this disparity is widened when yeasts, moulds, protozoa and algae are considered. The underlying reasons for these varied responses are poorly understood at present, but the chemical composition of outer cellular layers is likely to be a factor of prime importance. Other possible contributory factors may be differences in stress responses, the presence of efflux pumps and cells occurring within biofilms or algal mats.

Synopsis: It is difficult to predict accurately the ultimate effectiveness of a preservative in any but the simplest cosmetic or pharmaceutical formulation. It is thus necessary to obtain some assurance of its likely in‐use performance in a formulated product. A challenge test is a procedure in which a product is challenged by exposure to specified types of bacteria and fungi to determine whether it is adequately preserved. Assessment of preservative efficacy is needed over the intended shelf‐life of that product. Test organisms should be representative of those likely to occur as contaminants during use and should consist of Gram‐positive and Gram‐negative bacteria, mould and yeast. In‐house factory isolates obtained as a result of contamination of earlier batches of a product may also be included. The organisms, as single or mixed inocula, are inoculated (usually as a single challenge, although some advocate multiple challenges) into samples of the product and aliquots removed at appropriate intervals for the determination of survivors. Interpretation of data is normally based on pharmacopoeial or other official protocols. Challenge testing should be undertaken at the beginning, during and at the end of the shelf‐life of the product. An alternative, the D‐value, approach is open to criticism and further studies are required that utilize rapid methods, e.g. impedance, for the detection of survivors before these can be considered to be a suitable replacement for the more traditional but very time‐consuming, viable counting procedures.

1Welsh School of Pharmacy and 2School of Molecular and Medical Biosciences, Cardiff University, Cardiff CF10 3XF, UK

Objectives: Polyquad (Alcon) (polyquaternium-1, PQ-1) and Aldox (Alcon) (myristamidopropyl dimethylamine, MAPD) are two biocides that are used commercially in a contact lens disinfecting solution, namely Opti-Free Express (Alcon) multi-purpose disinfecting solution. Their potential mechanisms of action were investigated against a range of common ocular pathogens. These were Acanthamoeba castellanii (trophozoites and cysts), Aspergillus fumigatus, Candida albicans, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus. Methods: Three aspects were investigated: the lethal effects of the biocides on the organisms, the leakage of K⁺ from treated cells, and the lysis of spheroplasts derived from the cells. Results: PQ-1 was found to have predominantly antibacterial activity, and induced K⁺ leakage from the bacteria and C. albicans. It also caused lysis of spheroplasts of S. marcescens, but not those of C. albicans. MAPD was active against all of the organisms, but showed higher activity against the fungi and amoeba. It induced K⁺ leakage from A. fumigatus and C. albicans, and like PQ-1, lysed the spheroplasts of S. marcescens but not C. albicans. Conclusions: The two biocides have different spectra of antimicrobial activity. PQ-1 has mainly antibacterial activity, whereas MAPD was active against all of the test organisms, particularly the fungi.

High temperatures have profound effects on the structural and physiological properties of sporulating and non-sporulating bacteria, with membranes, RNA, DNA, ribosomes, protein and enzymes all affected. Nevertheless, it is apparent that no one single event is responsible for cell death. The induction of intracellular heat-shock proteins and the activation of extracellular alarmones in vegetative cells exposed to mildly lethal temperatures are important cell responses. In bacterial spores, several factors contribute to the overall resistance to moist (wet) and dry heat; the latter, but not the former, induces mutations. Heat resistance develops during sporulation, when spore-specific heat-shock proteins are also produced. Heat sensitivity is regained during germination of spores.

The mechanisms of the mycobactericidal action of ortho-phthalaldehyde (OPA), glutaraldehyde (GTA) and chlorhexidine diacetate (CHA) were investigated using mycobacterial spheroplasts of two reference strains, Mycobacterium chelonae NCTC 946, Mycobacterium abscessus NCTC 10882 and two GTA-resistant strains, M. chelonae Epping and M. chelonae Harefield. Transmission electron microscopy of the spheroplasts revealed an altered cell wall structure compared with the parent cells. Structural alterations resulting from the spheroplasting process were in part correlated to a loss of lipid content. Low concentrations of CHA induced protein coagulation in M. chelonae NCTC 946 spheroplasts, which also exhibited the highest loss of free non-polar lipids. Higher concentrations of CHA were required to produce similar results to the other spheroplasts investigated in which there was a less substantial decrease in lipid content. OPA (0.5% w/v) readily penetrated the residual cell wall and cytoplasmic membrane, producing significant protein coagulation in M. chelonae NCTC 946. GTA (0.5% v/v) induced a similar effect but to a lesser extent. Pre-treatment of the spheroplasts with OPA and GTA and their subsequent suspension in water demonstrated that GTA was a more potent cross-linking agent. This protective effect of GTA results from extensive cross-linking of amino and/or sulphydryl side-chain groups of proteins. The rapid mycobactericidal effect of OPA probably arises from its more efficient penetration across biological membranes. Mycobacterial spheroplasts represented a useful cellular model with an altered cell wall permeability. This study also showed the importance of the mycobacterial cell wall in conferring intrinsic resistance to CHA.

Potential development of bacterial tolerance to biocides in the industrial environment is examined in this study. Bacteria tolerant to the phenolic-type agent para-chloro-meta-xylenol (PCMX) and the bis-phenol 2,4,4′-trichloro-2′-hydroxydiphenylether (triclosan) were isolated from industrial sources and identified. Minimum inhibitory concentrations (MICs) were determined and compared with those of culture collection (standard) strains. Of around 100 isolates originally obtained, most were naturally tolerant species such as Pseudomonas spp., or showed low tolerance levels. PCMX-tolerant isolates of Pseudomonas stutzeri and triclosan-tolerant isolates of Citrobacter freundii and Acinetobacter johnsonii were retained for further study. Of these, only P. stutzeri and A. johnsonii showed elevated tolerance compared with the standard strains. There was no evidence of tolerance to the other biocide except for Pseudomonas aeruginosa (an intrinsically tolerant microorganism), and tolerances were stable in the absence of selective pressure except for A. johnsonii. Attempts to select or generate increased tolerance in the standard strains were unsuccessful. High tolerances in terms of MIC were not reflected in terms of lethal effects. This study did not produce any evidence suggesting that the presence of residual biocide concentrations in the industrial environment promotes the emergence of bacterial tolerance for them.