Aims: This study investigates the antimicrobial activity and mode of action of two natural products, eugenol and thymol, a commonly utilized biostatic agent, triclocarban (TCC), and two surfactants, didecyldimethylammonium chloride (DDDMAC) and C 10 -C 16 alkyldimethyl amine N-oxides (ADMAO). Methods and Results: Methods used included: determination of minimum inhibitory concentrations (MICs), lethal effect studies with suspension tests and the investigation of sub-MIC concentrations on growth of E. coli, Staph. aureus and Ps. aeruginosa using a Bioscreen microbiological analyser. Leakage of intracellular constituents and the effects of potentiating agents were also investigated. Only DDDMAC was bactericidal against all of the organisms tested. Eugenol, thymol and ADMAO showed bacteriostatic and bactericidal activity, but not against Ps. aeruginosa. TCC was only bacteristatic against Staph. aureus, but like the other agents, it did affect the growth of the other organisms in the Bioscreen experiments. All of the antimicrobial agents tested were potentiated by the permeabilizers to some extent and leakage of potassium was seen with all of the agents except TCC. Conclusions: DDDMAC was bactericidal against all organisms tested and all compounds had some bacteriostatic action. Low level static effects on bacterial growth were seen with sub-MIC concentrations. Membrane damage may account for at least part of the mode of action of thymol, eugenol, DDDMAC and ADMAO. Significance and Impact of the Study: The ingredients evaluated demonstrated a range of bactericidal and bacteriostatic properties against the Gram-negative and -positive organisms evaluated and the membrane (leakage of intracellular components) was implicated in the mode of action for most (except TCC). Sub-MIC levels of all ingredients did induce subtle effects on the organisms which impacted bacterial growth, even for those which had no true inhibitory effects.

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log 10 cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log 10 cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.Clinical epidemiologists have long recognized the potential of sink drains in hospital wards to harbor pathogens. Several studies have identified sink drains within medical-surgical intensive-care wards (19,22,30,35) and cystic fibrosis units (24) as possible sources of infection. Despite the increased information relating to the occurrence of bacterial biofilms and their reported involvement in the biofouling of domestic drains (7), there are few reports in the literature concerning the ecology and microbiology of this environment. The persistence (1) and significance (10) of biofilms in virtually all environments is widely acknowledged. Studies in the home (14, 34) have identified the potential health risks of microbial contamination. Scott et al. (34) identified possible pathogens in the kitchen, toilet, and bathrooms in Ͼ200 homes in the United Kingdom. More recent studies (3,8,9) have demonstrated that homes represent an environment into which bacterial, viral, and fungal pathogens are continuously introduced in association with food, people, and pets. Studies by Cogan et al. (8,9) showed that detergent-based cleaning was relatively ineffective in controlling the spread of salmonella and campylobacter to kitchen surfaces during the preparation of contaminated poultry. Despite such concerns, there have been few investigations into the bacterial composition of biofilms within domestic sink drains. As with hospital drains, the pipe work presents a variety of solid surfaces that are suitable substrates for biofilm formation (7, 26). Biofilm has been implicated in a high proportion of slow-running drains in the United States (7). Domestic drains are subject to intermittent wetting, periodic feeding with a plet...

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Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log 10 CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCRdenaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.Chlorhexidine (CHX), a cationic bis-biguanide biocide with low mammalian toxicity and broad-spectrum antibacterial (6) activity, was first described in 1954 (5). The primary mechanism of action of this biocide is membrane disruption, causing concentration-dependent growth inhibition and cell death (18). Secondary interactions causing inhibition of proteolytic and glycosidic enzymes may also be significant (15). With respect to dental hygiene applications, the cationic nature of CHX enables it to bind to tooth surfaces and oral mucosa, reducing pellicle formation and increasing substantivity through controlled release of the agent (2). The efficacy of CHX in reducing oral bacterial viability (14,36,42), strongly inhibiting plaque regrowth, and preventing gingivitis (25) has been demonstrated in many studies (7). Relatively few investigations have considered longer-term effects of CHX use. An early study, however, demonstrated that oral treatment of human volunteers with CHX resulted in a 30 to 50% reduction in total bacterial counts with an associated reduction in counts of Streptococcus mutans (38).Recent reports have demonstrated that the chlorinated diphenylether antibacterial triclosan (TCS) can select for mutants in the FabI gene of Escherichia coli at sublethal co...