Ethylzingerone, a Novel Compound with Antifungal Activity.

Preservatives increase the shelf life of cosmetic products by preventing growth of contaminating microbes, including bacteria and fungi. In recent years, the Scientific Committee on Consumer Safety (SCCS) has recommended the ban or restricted use of a number of preservatives due to safety concerns. Here, we characterize the antifungal activity of ethylzingerone (hydroxyethoxyphenyl butanone [HEPB]), an SCCS-approved new preservative for use in rinse-off, oral care, and leave-on cosmetic products. We show that HEPB significantly inhibits growth of Candida albicans, Candida glabrata, and Saccharomyces cerevisiae, acting fungicidally against C. albicans Using transcript profiling experiments, we found that the C. albicans transcriptome responded to HEPB exposure by increasing the expression of genes involved in amino acid biosynthesis while activating pathways involved in chemical detoxification/oxidative stress response. Comparative analyses revealed that C. albicans phenotypic and transcriptomic responses to HEPB treatment were distinguishable from those of two widely used preservatives, triclosan and methylparaben. Chemogenomic analyses, using a barcoded S. cerevisiae nonessential mutant library, revealed that HEPB antifungal activity strongly interfered with the biosynthesis of aromatic amino acids. The trp1Δ mutants in S. cerevisiae and C. albicans were particularly sensitive to HEPB treatment, a phenotype rescued by exogenous addition of tryptophan to the growth medium, providing a direct link between HEPB mode of action and tryptophan availability. Collectively, our study sheds light on the antifungal activity of HEPB, a new molecule with safe properties for use as a preservative in the cosmetic industry, and exemplifies the powerful use of functional genomics to illuminate the mode of action of antimicrobial agents.

Mapping the Efficacy and Mode of Action of Ethylzingerone [4-(3-Ethoxy-4-Hydroxyphenyl) Butan-2-One] as an Active Agent against Burkholderia Bacteria.

Burkholderia cepacia complex (Bcc) bacteria are intrinsically antimicrobial-resistant opportunistic pathogens and key risk species in the contamination of nonfood industrial products. New agents and formulations to prevent growth of Burkholderia in home care (cleaning agents) and personal-care (cosmetics and toiletries) products are required. We characterized how ethylzingerone [4-(3-ethoxy-4-hydroxyphenyl) butan-2-one] (HEPB) acts as a preservative with activity against Burkholderia species encountered in industry. Burkholderia (n = 58) and non-Burkholderia (n = 7) bacteria were screened for susceptibility to HEPB, and its mode of action and resistance were determined for a model Burkholderia vietnamiensis strain using transposon mutagenesis, transcriptomics, and genome resequencing analysis. The susceptibility of Burkholderia spp. to HEPB (MIC = 0.45% ± 0.11% [wt/vol]; MBC = 0.90% ± 0.3% [wt/vol]) was characterized, with limited inter- and intraspecies differences. HEPB (1% [wt/vol]) was rapidly bactericidal, producing a 6-log reduction in viability within 4 h. Spontaneous resistance to HEPB did not develop, but transient phenotypes with altered growth characteristics and susceptibility to antibiotics were identified after prolonged exposure to sublethal HEPB concentrations. Transposon mutagenesis and RNA-sequencing analysis identified multiple genetic pathways associated with HEPB exposure, including stress response mechanisms, altered permeability, regulation of intracellular pH, damage and repair of intracellular components, and alteration and repair of lipopolysaccharides. Key pathways included the stringent response, homeostasis of intracellular pH by the kdp operon, protection against electrophiles by KefC, and repair of oxidized proteins by methionine sulfoxide reductase enzymes. In summary, we show that HEPB has potent, targeted efficacy against Burkholderia bacteria without promoting wider stable antimicrobial resistance. The mode of action of HEPB against Burkholderia is multifactorial, but killing by intracellular oxidation is a key mechanism of this promising agent.IMPORTANCEBurkholderia bacteria are opportunistic pathogens that can overcome preservatives used in the manufacture of nonsterile industrial products and occasionally cause contamination. Consequently, new preservatives to prevent the growth of key risk Burkholderia cepacia complex bacteria in nonfood industrial products are urgently required. Here, we show that ethylzingerone is active against these problematic bacteria, killing them via a multifactorial mode of action which involves intracellular oxidation.

Hydroxyethoxy phenyl butanone, a new cosmetic preservative, does not cause bacterial cross-resistance to antimicrobials.

Introduction. Biocide-induced cross-resistance to antimicrobials in bacteria has been described and is a concern for regulators. We have recently reported on a new protocol to predict the propensity of biocide to induce phenotypic resistance in bacteria.Aim. To measure bacterial propensity to develop antimicrobial resistance following exposure to a new cosmetic preservative developed by L'Oréal R and I.Methodology. Well-established antimicrobials including triclosan (TRI) and benzalkonium chloride (BZC) and a new molecule hydroxyethoxy phenyl butanone (HEPB) were investigated for their antimicrobial efficacy, effect on bacterial growth, and their potential to induce resistance to chemotherapeutic antibiotics using a new predictive protocol.Results. The use of this predictive protocol with Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa showed that TRI and BZC significantly affected bacterial growth, MICs and minimum bactericidal concentrations (MBCs). There was no change in antibiotic susceptibility profile following exposure to BZC, but E. coli became intermediate resistant to tobramycin following treatment with TRI (0.00002 % w/v). HEPB did not change the antimicrobial susceptibility profile in P. aeruginosa and S. aureus but E. coli became susceptible to gentamicin. TRI exposure resulted in bacterial susceptibility profile alteration consistent with the literature and confirmed the use of TRI as a positive control in such a test.Conclusion. Data produced on the propensity of a molecule to induce bacterial resistance is useful and appropriate when launching a new preservative.

Dandruff is associated with disequilibrium in the proportion of the major bacterial and fungal populations colonizing the scalp.

The bacterial and fungal communities associated with dandruff were investigated using culture-independent methodologies in the French subjects. The major bacterial and fungal species inhabiting the scalp subject's were identified by cloning and sequencing of the conserved ribosomal unit regions (16S for bacterial and 28S-ITS for fungal) and were further quantified by quantitative PCR. The two main bacterial species found on the scalp surface were Propionibacterium acnes and Staphylococcus epidermidis, while Malassezia restricta was the main fungal inhabitant. Dandruff was correlated with a higher incidence of M. restricta and S. epidermidis and a lower incidence of P. acnes compared to the control population (p<0.05). These results suggested for the first time using molecular methods, that dandruff is linked to the balance between bacteria and fungi of the host scalp surface.

Efficiency of water disinfectants against Legionella pneumophila and Acanthamoeba.

Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment. With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chlorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems.

Assessment of the removal and inactivation of influenza viruses H5N1 and H1N1 by drinking water treatment.

Since 2003, there has been significant concern about the possibility of an outbreak of avian influenza virus subtype H5N1. Moreover, in the last few months, a pandemic of a novel swine-origin influenza A virus, namely A(H1N1), has already caused hundreds of thousands of human cases of illness and thousands of deaths. As those viruses could possibly contaminate water resources through wild birds excreta or through sewage, the aim of our work was to find out whether the treatment processes in use in the drinking water industry are suitable for eradicating them. The effectiveness of physical treatments (coagulation-flocculation-settling, membrane ultrafiltration and ultraviolet) was assessed on H5N1, and that of disinfectants (monochloramine, chlorine dioxide, chlorine, and ozone) was established for both the H5N1 and H1N1 viruses. Natural water samples were spiked with human H5N1/H1N1 viruses. For the coagulation-settling experiments, raw surface water was treated in jar-test pilots with 3 different coagulating agents (aluminum sulfate, ferric chloride, aluminum polychorosulfate). Membrane performance was quantified using a hollow-fiber ultrafiltration system. Ultraviolet irradiation experiments were conducted with a collimated beam that made it possible to assess the effectiveness of various UV doses (25-60 mJ/cm2). In the case of ozone, 0.5 mg/L and 1 mg/L residual concentrations were tested with a contact time of 10 min. Finally, for chlorine, chlorine dioxide and monochloramine treatments, several residual oxidant target levels were tested (from 0.3 to 3 mg/L) with contact times of 5-120 min. The infectivity of the H5N1 and H1N1 viruses in water samples was quantified in cell culture using a microtiter endpoint titration. The impact of coagulation-settling on the H5N1 subtype was quite low and variable. In contrast, ultrafiltration achieved more than a 3-log reduction (and more than a 4-log removal in most cases), and UV treatment was readily effective on its inactivation (more than a 5-log inactivation with a UV dose of 25 mJ/cm2). Of the chemical disinfection treatments, ozone, chlorine and chlorine dioxide were all very effective in inactivating H5N1 and H1N1, whereas monochloramine treatment required higher doses and longer contact times to achieve significant reductions. Our findings suggest that the water treatment strategies that are currently used for surface water treatment are entirely suitable for removing and/or inactivating influenza A viruses. Appropriate preventive actions can be defined for single disinfection treatment plants.

Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water.

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.