No abstract

No abstract

Microemulsions are physically stable oil/water systems that have potential use as delivery systems for many pharmaceuticals which are normally of limited use due to their hydrophobicity, toxicity or inability to access the site of action. It has been suggested that microemulsions are self‐preserving antimicrobials in their own right, although there is little evidence to support this. In this experiment, microemulsions of various compositions were formulated and tested for their stability and antimicrobial action. The physical stability of the different microemulsions was assessed by centrifugation at 4000 g and by storage in a water bath at 37 °C for one month, during which no phase separation was observed. The antimicrobial activity of the microemulsions was tested using the compendial method, observation of the kinetics of killing, and transmission electron microscopy (TEM) of microemulsion‐exposed cultures of Pseudomonas aeruginosa PA01. These latter experiments on Ps. aeruginosa indicated distinct signs of membrane disruption. The results indicated that the microemulsions are self‐preserved, and that their killing of microbial cultures is very rapid and may be the result of membrane activity.

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1x MIC) gave 99.99%o reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.

Benzisothiazolone (BIT), N‐methylisothiazolone (MIT) and 5‐chloro‐N‐methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub‐minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol‐interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T‐OMP, in SDS‐PAGE preparations. T‐OMP was observed to disappear from the biocide‐exposed preparations and reappear when the resistance‐induced cultures were passaged in the absence of biocide. This reappearance of T‐OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross‐resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T‐OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon™ form (CMIT and MIT).

LI E R. 1998. Sodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively) are widely used as cosmetic preservatives and general antimicrobial agents. They have been shown to be active against fungal cell walls, associated membranes and bacterial transport processes. Investigations were undertaken into the effect of these antimicrobial agents on substrate catabolism and intracellular ATP levels using an oxygen electrode and luciferin-luciferase technology, respectively. Results indicate that, while both compounds are poor inhibitors of substrate catabolism, sub-inhibitory concentrations of biocide greatly reduces intracellular ATP levels in both Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. This is thought to be due to the action of NaPT and ZnPT on the Gram-negative bacterial membrane. INTRODUCTIONSodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively, Fig. 1) are the sodium salt and zinc chelate of 1-hydroxy-2-pyridinethiol (Shaw et al. 1950 ;Albert et al. 1956 ;Hyde and Nelson 1984). Both NaPT and ZnPT have been shown to possess a wide spectrum of antimicrobial activity against fungal and bacterial species (Pansy et al. 1953 ;Albert et al. 1956 ;Hyde and Nelson 1984 ;Khattar et al. 1988). They are widely used as cosmetic preservatives (Nelson and Hyde 1981 ;Hyde and Nelson 1984) and are incorporated as anti-dandruff agents in shampoos (Fredriksin and Feargeman 1983 ;Hyde and Nelson 1984). They also possess excellent metal chelating properties and ZnPT is active as a metal complex (Davies 1985 ;Fenn and Alexander 1988).Previous work has suggested that this group of compounds is membrane active. This is indicated by the inhibition of uptake of several unrelated substrates in both bacteria and fungi (Chandler and Segel 1978;Friedman 1981;Khattar et al. 1988 ;Khattar and Salt 1993) and the observed depolarization of the transmembrane electrical potential in Neurospora crassa (Ermolayeva et al. 1995). The effects of an antimicrobial agent on substrate transport and related metabolism may be used as indicators of the membrane activity of the test agent (Gilbert et al. 1991). In turn, these effects may be reflected as a reduction in intracellular ATP levels (Harold Dundee, Bell Street, Dundee, DD1 1HG,.© 1998 The Society for Applied Microbiology 1972). Regardless of substrate transport, a direct membrane effect by a biocide may decrease intracellular ATP levels via disruption of the transmembrane proton motive force (PMF) and related processes. This has been exhibited with several membrane active agents including fentichlor, PHMB, alexidine and chlorhexidine (Bloomfield 1974 ;Chopra et al. 1987 ;Chawner and Gilbert 1989).This study describes the activity of NaPT and ZnPT on substrate transport and catabolism in Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548 and the effects of these agents on bacterial intracellular ATP levels at sub-inhibitory and minimal inhibitory concentrations (MIC). MATERIALS AND METHODS Organisms and chemicalsStock cultures of Es...

The increasing use of microbicides in consumer products is raising concerns related to enhanced microbicide resistance in bacteria and potential cross resistance to antibiotics. The recently published documents on this topic from the European Commission have spawned much interest to better understand the true extent of the putative links for the benefit of the manufacturers, regulators, and consumers alike. This white paper is based on a 2-day workshop (SEAC-Unilever, Bedford, United Kingdom; June 2012) in the fields of microbicide usage and resistance. It identifies gaps in our knowledge and also makes specific recommendations for harmonization of key terms and refinement/standardization of methods for testing microbicide resistance to better assess the impact and possible links with cross resistance to antibiotics. It also calls for a better cohesion in research in this field. Such information is crucial to developing any risk assessment framework on microbicide use notably in consumer products. The article also identifies key research questions where there are inadequate data, which, if addressed, could promote improved knowledge and understanding to assess any related risks for consumer and environmental safety.