Medical Mycology, 2015, 53, 396–404 doi: 10.1093/mmy/myv006 Original Article
Original Article
Efficacy of Ethanol against Trichosporon asahii Biofilm in vitro Yong Liao1,† , Hui Zhao1,2,† , Xuelian Lu1 , Suteng Yang1 , Jianfeng Zhou1 and Rongya Yang1,∗ 1
Department of Dermatology, General Hospital of Beijing Military Command, Beijing 100700, China and Department of Dermatology, Tongzhou Maternal and Child Health Hospital of Beijing, Beijing 101101, China
2
†
Both authors contributed equally to this work.
Received 25 September 2014; Revised 13 December 2014; Accepted 17 January 2015
Abstract Trichosporon asahii (T. asahii) can cause invasive infections, particularly catheter-related bloodstream infections (CR-BSIs). T. asahii biofilm, which is resistant to the most common clinical antifungal agents, may play an important role in these life-threatening infections. This study focused on the effects of ethanol on the different phases of T. asahii biofilm formation. At the concentrations clinically used, ethanol killed T. asahii planktonic cells (MIC90 = 15% and m-MIC90 = 15%) and biofilm (SMIC90 = 50%), and exposure to 25% ethanol for 12 h or to 50% ethanol for 8 h completely inhibited biofilm development and eradicated mature T. asahii biofilm. Thus, our results showed that ethanol effectively inhibited the main phases of T. asahii biofilm formation. This study reveals a new potential strategy to prevent and treat T. asahii biofilm-related CR-BSIs. Key words: Trichosporon asahii, biofilm, ethanol.
Introduction Trichosporon spp. belong to a group of basidiomycete yeast-like fungi that are widely distributed in nature and occasionally occur as normal human flora of the skin, gastrointestinal tract, and respiratory tract [1]. Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening invasive infections in immunocompromised hosts, especially in patients with hematological malignant diseases (HMDs) [2]. Invasive trichosporonosis is often associated with fungemia (58.8–74.7%) [2–4], and Trichosporon spp. is the second most common pathogen of yeast fungemia in patients with HMDs [5]. It has been 396
reported that most patients with Trichosporon fungemia (TF) have received a central venous catheter (CVC) before the onset of infection, the majority of which are catheterrelated bloodstream infections (CR-BSIs) (70%) [3,6]. Although received the antifungal therapy, the mortality of patients with TF remains high (53–76%) [3,6]. As the major cause of TF, T. asahii has been found in an in vitro study to form a biofilm on 35-mm-diameter polystyrene tissue culture dishes that develops in the following four phases: adhesion (0 to 2 h), germination and microcolony formation (2 to 4 h), filamentation (4 to 6 h), and proliferation and maturation (24 to 72 h) [7]. The
C The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://mmy.oxfordjournals.org/ at Seton Hall on April 28, 2015
*To whom correspondence should be addressed. Rongya Yang, Department of Dermatology, General Hospital of Beijing Military Command, No.5, Nanmencang St., East District, Beijing 100700, China. Tel/Fax: +86-10-66721229; E-mail: [email protected]
Liao et al.
Materials and methods Strains and clinical sources Seven clinical strains of T. asahii evaluated in this study were previously isolated from blood samples (five strains) and blood catheter tips (two strains) from patients with T. asahii CR-BSIs. All clinical strains were identified using a commercial system (API 20C AUX, BioM´erieux, France) and confirmed via DNA sequencing of the intergenic spacer 1 (IGS1) region of the rRNA gene, as previously reported [16]. The type strain of T. asahii (CBS2479) was purchased from the CBS-KNAW Fungal Biodiversity Centre (the Netherlands). All strains were stored as frozen stocks at −80◦ C at the Laboratory for Medical Mycology of the General Hospital of Beijing Military Command until use. The strains were subcultured on Sabouraud dextrose agar (SDA, Merck KGaA, Darmstadt, Germany) at 37◦ C. A single, freshly grown colony was selected and cultured overnight in yeast peptone dextrose (YPD, Oxoid Limited, England)
liquid medium at 37◦ C on an orbital shaker (130 rpm). Following growth, the cells were harvested by centrifugation and washed twice with sterile phosphate-buffered saline (PBS). The cells were resuspended in RPMI 1640 (SigmaAldrich China, Shanghai, China) adjusted to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (SigmaAldrich China) to densities of 103 CFU/ml and 106 CFU/ml for the planktonic cell study and 106 CFU/ml for the biofilm study, as counted with a hemocytometer.
Quantitation assay of planktonic cells and biofilm The sodium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]- 2H-tetrazolium hydroxide (XTT) (Sigma-Aldrich China) reduction assay was used to determine the metabolic activities of T. asahii planktonic cells and biofilm as previously reported [7,8]. XTT was prepared in a saturated solution at 0.5 g/l in sterile Ringer’s lactate. The XTT solution was sterilized through a 0.22-μm R pore-size filter (MillexGP, USA). A 10-mM stock solution of menadione (Sigma-Aldrich China) in 100% acetone (Sigma-Aldrich China) was prepared and then added to the XTT solution at a final concentration of 1 μM. After adding 100 μl of the XTT/menadione solution to each well, the plate was incubated in the dark for 2 h at 37◦ C. Supernatants were centrifuged, and 80 μl from each well was transferred into the wells of a new microtiter plate. These plates were then read at 492 nm with a Multiskan MK3 microplate reader (Thermo Fisher Scientific Inc., USA).
Antifungal susceptibility testing of planktonic cells The in vitro activity of ethanol against planktonic cells was determined using a M27-A3 broth microdilution method (the Clinical and Laboratory Standards Institute, CLSI, USA) and XTT reduction assay. The standard CLSI inocula (103 CFU/ml in RPMI 1640) and modified inocula (106 CFU/ml in RPMI 1640) were grown in 96-well polystyrene plates, and the ethanol was diluted in RPMI 1640-MOPS medium. The wells containing RPMI 1640-MOPS medium served as background controls. The final working concentrations of ethanol were 0.5%, 1%, 2%, 4%, 8%, 15%, 25%, and 50%. The end-points for minimal inhibitory concentration (MIC) and modified minimal inhibitory concentration (m-MIC) were defined as the lowest concentration of ethanol that caused a 100% reduction in metabolic activity compared to the growth controls.
Biofilm formation A volume of 100 μl of the adjusted T. asahii suspension (106 CFU/ml) was added to 96-well microtiter plates. The wells
Downloaded from http://mmy.oxfordjournals.org/ at Seton Hall on April 28, 2015
T. asahii biofilm is instinctually resistant to antifungal agents, including itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin [7,8], and even decreased SMICs of the voriconazole/amphotericin B combination are still too high for clinical usage [9]. As an important virulence factor, biofilm formation may be associated with T. asahii CR-BSIs [1,7], aiding in pathogen escape from antifungal drugs and the host immune system [10]. Fungal CR-BSIs, which are biofilm-related infections, are often difficult to treat and eradicate, leading to persistent or recurrent infections with high mortality [11]. Therefore, it is necessary to find a more effective strategy for their prevention and treatment. Antifungal lock therapy (AFLT) for the purpose of catheter salvage has been studied in vitro and in animal models and utilized in the management of Candida CR-BSIs [12]. AFLT with standard antifungal agents may lead to the development of resistance. Ethanol is mainly used as an antiseptic in medical practice to promote membrane damage, the denaturation of proteins, the dissolving of lipids, and subsequent interference with the metabolism and lysis of pathogen cells [13]. Ethanol can effectively inhibit the biofilm formation of Candida albicans (C. albicans) and other bacteria in vitro and can be used in ethanol lock therapy (ELT) for both the prevention and treatment of CR-BSIs [14,15]. However, there has been no study systematically examining the effects of ethanol concentration and dwell time on T. asahii biofilm formation. The purpose of this study was to determine the optimal concentration and dwell time of ethanol for the effective inhibition of the main phases of T. asahii biofilm formation.
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containing RPMI 1640-MOPS medium without T. asahii served as background controls. After 1 h of incubation (adhesion phase) at 37◦ C, each well was washed twice gently with sterile PBS to remove non-adherent cells and then refilled with 200 μl of fresh RPMI 1640-MOPS medium for further incubation at 37◦ C.
Antifungal susceptibility testing of sessile (biofilm) cells
The effect of ethanol on the adhesion phase of T. asahii biofilm One hundred microliters of suspension (106 CFU/ml) was added to each well of the 96-well microtiter plates. Ethanol at final working concentrations of 0.125%, 0.25%, 1%, 2%, and 4% was added to each experimental well at the same time, and cells were allowed to adhere for various lengths of time (0.5 h, 1 h, and 2 h) at 37◦ C. Wells without ethanol were used as growth controls. Supernatants were aspirated, and non-adherent cells were washed twice gently with sterile PBS. Then, the quantity of adherent cells in each well was measured via an XTT reduction assay. Compared to the growth control, the percent change in metabolic activity of each experimental well was calculated for further analysis.
The effect of ethanol on the germination phase of T. asahii biofilm The washed cells were harvested as previously described and then filtered with 4 layers of sterile medical gauze to obtain >90% yeast cell suspensions. After counting with a hemocytometer, a final suspension was prepared at a density of 106 CFU/ml in RPMI 1640-MOPS medium. One hundred microliters of suspension (106 CFU/ml) was added to each well of the 96-well microtiter plates. After 1 h of incubation (adhesion phase) at 37◦ C, each well was washed twice gently with sterile PBS to remove nonadherent cells
and then ethanol in RPMI 1640-MOPS medium was added to each experimental well at the final working concentrations (0.125%, 0.25%, 1%, 2%, and 4%). Wells without ethanol were used as growth controls. After incubation for various lengths of time (0.5 h, 1 h, 2 h, 4 h, and 8 h) at 37◦ C, microscopic observations of the cells were performed. The effect of ethanol was quantified by counting the number of individual yeast and germ tubes formed in the population. More than 100 cells were counted for each well. The percentage of germ tube formation in each well was calculated as previously described [17].
The effect of ethanol on the formation and mature phase of T. asahii biofilm After 1 h of incubation (adhesion phase) at 37◦ C, each well was washed twice gently with sterile PBS to remove non-adherent cells as described previously. Then, 200 μl of RPMI 1640-MOPS medium with various concentrations (0.125%, 0.25%, 0.5%, 1%, 2%, 4%, 8%, 15%, 25%, and 50%) of ethanol was added to each experimental well. After incubation for various lengths of time (2 h, 4 h, 8 h, 12 h, and 24 h) at 37◦ C, the quantity of biofilm in each well was measured via an XTT reduction assay. Biofilm formation was performed as previously described. After 48 h of incubation at 37◦ C, the mature T. asahii biofilms were washed twice with PBS, and various concentrations (1%, 2%, 4%, 8%, 15%, 25%, and 50%) of ethanol in 200 μl RPMI 1640-MOPS medium were added to each experimental well. After incubation for various lengths of time (0.5 h, 1 h, 2 h, 4 h, and 12 h) at 37◦ C, the quantity of biofilm in each well was also measured via an XTT reduction assay. The metabolic activity of the biofilm in each experimental well was assessed and compared to the growth control for further analysis.
Statistical analysis All experiments were repeated at least two times, and the standard deviation from the mean of each tested strain was calculated. The effects of ethanol on the different phases of T. asahii biofilm were analyzed using one-way and two-way analysis of variance (ANOVA) with SPSS 16.0 (Statistical Product and Service Solutions, IBM SPSS, USA). A P50% T. asahii adhesion in all strains.
The effect of ethanol on the germination phase of T. asahii biofilm The effects of ethanol on the germination phase of T. asahii were concentration-dependent (Fig. 2). Lower concentrations (0.125% and 0.25%) of ethanol significantly induced
Inhibitory effects of ethanol on the formation and mature phases of T. asahii biofilm After T. asahii cell adhesion, the addition of ethanol suppressed the normal development of T. asahii biofilm. Ethanol demonstrated inhibitory activity against biofilm formation in concentration- and time-dependent manners when it was present at a concentration of 2% or higher, with increasing concentrations and incubation times resulting in the reduced metabolic activity of the T. asahii cells (Fig. 3). Further, exposure to 25% ethanol for 8 h or 50% ethanol for 4 h inhibited T. asahii biofilm formation by 99.15% and 99.03%, respectively. After 48 h, corresponding to the formation of the mature biofilm of T. asahii (the late phase of biofilm formation), only ethanol concentrations of 15% or higher inhibited the metabolic
Figure 1. Inhibitive effects of ethanol at the adhesion phase of T. asahii biofilm. Error bars represent SDs. ∗ means P
Original Article
Efficacy of Ethanol against Trichosporon asahii Biofilm in vitro Yong Liao1,† , Hui Zhao1,2,† , Xuelian Lu1 , Suteng Yang1 , Jianfeng Zhou1 and Rongya Yang1,∗ 1
Department of Dermatology, General Hospital of Beijing Military Command, Beijing 100700, China and Department of Dermatology, Tongzhou Maternal and Child Health Hospital of Beijing, Beijing 101101, China
2
†
Both authors contributed equally to this work.
Received 25 September 2014; Revised 13 December 2014; Accepted 17 January 2015
Abstract Trichosporon asahii (T. asahii) can cause invasive infections, particularly catheter-related bloodstream infections (CR-BSIs). T. asahii biofilm, which is resistant to the most common clinical antifungal agents, may play an important role in these life-threatening infections. This study focused on the effects of ethanol on the different phases of T. asahii biofilm formation. At the concentrations clinically used, ethanol killed T. asahii planktonic cells (MIC90 = 15% and m-MIC90 = 15%) and biofilm (SMIC90 = 50%), and exposure to 25% ethanol for 12 h or to 50% ethanol for 8 h completely inhibited biofilm development and eradicated mature T. asahii biofilm. Thus, our results showed that ethanol effectively inhibited the main phases of T. asahii biofilm formation. This study reveals a new potential strategy to prevent and treat T. asahii biofilm-related CR-BSIs. Key words: Trichosporon asahii, biofilm, ethanol.
Introduction Trichosporon spp. belong to a group of basidiomycete yeast-like fungi that are widely distributed in nature and occasionally occur as normal human flora of the skin, gastrointestinal tract, and respiratory tract [1]. Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening invasive infections in immunocompromised hosts, especially in patients with hematological malignant diseases (HMDs) [2]. Invasive trichosporonosis is often associated with fungemia (58.8–74.7%) [2–4], and Trichosporon spp. is the second most common pathogen of yeast fungemia in patients with HMDs [5]. It has been 396
reported that most patients with Trichosporon fungemia (TF) have received a central venous catheter (CVC) before the onset of infection, the majority of which are catheterrelated bloodstream infections (CR-BSIs) (70%) [3,6]. Although received the antifungal therapy, the mortality of patients with TF remains high (53–76%) [3,6]. As the major cause of TF, T. asahii has been found in an in vitro study to form a biofilm on 35-mm-diameter polystyrene tissue culture dishes that develops in the following four phases: adhesion (0 to 2 h), germination and microcolony formation (2 to 4 h), filamentation (4 to 6 h), and proliferation and maturation (24 to 72 h) [7]. The
C The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://mmy.oxfordjournals.org/ at Seton Hall on April 28, 2015
*To whom correspondence should be addressed. Rongya Yang, Department of Dermatology, General Hospital of Beijing Military Command, No.5, Nanmencang St., East District, Beijing 100700, China. Tel/Fax: +86-10-66721229; E-mail: [email protected]
Liao et al.
Materials and methods Strains and clinical sources Seven clinical strains of T. asahii evaluated in this study were previously isolated from blood samples (five strains) and blood catheter tips (two strains) from patients with T. asahii CR-BSIs. All clinical strains were identified using a commercial system (API 20C AUX, BioM´erieux, France) and confirmed via DNA sequencing of the intergenic spacer 1 (IGS1) region of the rRNA gene, as previously reported [16]. The type strain of T. asahii (CBS2479) was purchased from the CBS-KNAW Fungal Biodiversity Centre (the Netherlands). All strains were stored as frozen stocks at −80◦ C at the Laboratory for Medical Mycology of the General Hospital of Beijing Military Command until use. The strains were subcultured on Sabouraud dextrose agar (SDA, Merck KGaA, Darmstadt, Germany) at 37◦ C. A single, freshly grown colony was selected and cultured overnight in yeast peptone dextrose (YPD, Oxoid Limited, England)
liquid medium at 37◦ C on an orbital shaker (130 rpm). Following growth, the cells were harvested by centrifugation and washed twice with sterile phosphate-buffered saline (PBS). The cells were resuspended in RPMI 1640 (SigmaAldrich China, Shanghai, China) adjusted to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (SigmaAldrich China) to densities of 103 CFU/ml and 106 CFU/ml for the planktonic cell study and 106 CFU/ml for the biofilm study, as counted with a hemocytometer.
Quantitation assay of planktonic cells and biofilm The sodium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]- 2H-tetrazolium hydroxide (XTT) (Sigma-Aldrich China) reduction assay was used to determine the metabolic activities of T. asahii planktonic cells and biofilm as previously reported [7,8]. XTT was prepared in a saturated solution at 0.5 g/l in sterile Ringer’s lactate. The XTT solution was sterilized through a 0.22-μm R pore-size filter (MillexGP, USA). A 10-mM stock solution of menadione (Sigma-Aldrich China) in 100% acetone (Sigma-Aldrich China) was prepared and then added to the XTT solution at a final concentration of 1 μM. After adding 100 μl of the XTT/menadione solution to each well, the plate was incubated in the dark for 2 h at 37◦ C. Supernatants were centrifuged, and 80 μl from each well was transferred into the wells of a new microtiter plate. These plates were then read at 492 nm with a Multiskan MK3 microplate reader (Thermo Fisher Scientific Inc., USA).
Antifungal susceptibility testing of planktonic cells The in vitro activity of ethanol against planktonic cells was determined using a M27-A3 broth microdilution method (the Clinical and Laboratory Standards Institute, CLSI, USA) and XTT reduction assay. The standard CLSI inocula (103 CFU/ml in RPMI 1640) and modified inocula (106 CFU/ml in RPMI 1640) were grown in 96-well polystyrene plates, and the ethanol was diluted in RPMI 1640-MOPS medium. The wells containing RPMI 1640-MOPS medium served as background controls. The final working concentrations of ethanol were 0.5%, 1%, 2%, 4%, 8%, 15%, 25%, and 50%. The end-points for minimal inhibitory concentration (MIC) and modified minimal inhibitory concentration (m-MIC) were defined as the lowest concentration of ethanol that caused a 100% reduction in metabolic activity compared to the growth controls.
Biofilm formation A volume of 100 μl of the adjusted T. asahii suspension (106 CFU/ml) was added to 96-well microtiter plates. The wells
Downloaded from http://mmy.oxfordjournals.org/ at Seton Hall on April 28, 2015
T. asahii biofilm is instinctually resistant to antifungal agents, including itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin [7,8], and even decreased SMICs of the voriconazole/amphotericin B combination are still too high for clinical usage [9]. As an important virulence factor, biofilm formation may be associated with T. asahii CR-BSIs [1,7], aiding in pathogen escape from antifungal drugs and the host immune system [10]. Fungal CR-BSIs, which are biofilm-related infections, are often difficult to treat and eradicate, leading to persistent or recurrent infections with high mortality [11]. Therefore, it is necessary to find a more effective strategy for their prevention and treatment. Antifungal lock therapy (AFLT) for the purpose of catheter salvage has been studied in vitro and in animal models and utilized in the management of Candida CR-BSIs [12]. AFLT with standard antifungal agents may lead to the development of resistance. Ethanol is mainly used as an antiseptic in medical practice to promote membrane damage, the denaturation of proteins, the dissolving of lipids, and subsequent interference with the metabolism and lysis of pathogen cells [13]. Ethanol can effectively inhibit the biofilm formation of Candida albicans (C. albicans) and other bacteria in vitro and can be used in ethanol lock therapy (ELT) for both the prevention and treatment of CR-BSIs [14,15]. However, there has been no study systematically examining the effects of ethanol concentration and dwell time on T. asahii biofilm formation. The purpose of this study was to determine the optimal concentration and dwell time of ethanol for the effective inhibition of the main phases of T. asahii biofilm formation.
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containing RPMI 1640-MOPS medium without T. asahii served as background controls. After 1 h of incubation (adhesion phase) at 37◦ C, each well was washed twice gently with sterile PBS to remove non-adherent cells and then refilled with 200 μl of fresh RPMI 1640-MOPS medium for further incubation at 37◦ C.
Antifungal susceptibility testing of sessile (biofilm) cells
The effect of ethanol on the adhesion phase of T. asahii biofilm One hundred microliters of suspension (106 CFU/ml) was added to each well of the 96-well microtiter plates. Ethanol at final working concentrations of 0.125%, 0.25%, 1%, 2%, and 4% was added to each experimental well at the same time, and cells were allowed to adhere for various lengths of time (0.5 h, 1 h, and 2 h) at 37◦ C. Wells without ethanol were used as growth controls. Supernatants were aspirated, and non-adherent cells were washed twice gently with sterile PBS. Then, the quantity of adherent cells in each well was measured via an XTT reduction assay. Compared to the growth control, the percent change in metabolic activity of each experimental well was calculated for further analysis.
The effect of ethanol on the germination phase of T. asahii biofilm The washed cells were harvested as previously described and then filtered with 4 layers of sterile medical gauze to obtain >90% yeast cell suspensions. After counting with a hemocytometer, a final suspension was prepared at a density of 106 CFU/ml in RPMI 1640-MOPS medium. One hundred microliters of suspension (106 CFU/ml) was added to each well of the 96-well microtiter plates. After 1 h of incubation (adhesion phase) at 37◦ C, each well was washed twice gently with sterile PBS to remove nonadherent cells
and then ethanol in RPMI 1640-MOPS medium was added to each experimental well at the final working concentrations (0.125%, 0.25%, 1%, 2%, and 4%). Wells without ethanol were used as growth controls. After incubation for various lengths of time (0.5 h, 1 h, 2 h, 4 h, and 8 h) at 37◦ C, microscopic observations of the cells were performed. The effect of ethanol was quantified by counting the number of individual yeast and germ tubes formed in the population. More than 100 cells were counted for each well. The percentage of germ tube formation in each well was calculated as previously described [17].
The effect of ethanol on the formation and mature phase of T. asahii biofilm After 1 h of incubation (adhesion phase) at 37◦ C, each well was washed twice gently with sterile PBS to remove non-adherent cells as described previously. Then, 200 μl of RPMI 1640-MOPS medium with various concentrations (0.125%, 0.25%, 0.5%, 1%, 2%, 4%, 8%, 15%, 25%, and 50%) of ethanol was added to each experimental well. After incubation for various lengths of time (2 h, 4 h, 8 h, 12 h, and 24 h) at 37◦ C, the quantity of biofilm in each well was measured via an XTT reduction assay. Biofilm formation was performed as previously described. After 48 h of incubation at 37◦ C, the mature T. asahii biofilms were washed twice with PBS, and various concentrations (1%, 2%, 4%, 8%, 15%, 25%, and 50%) of ethanol in 200 μl RPMI 1640-MOPS medium were added to each experimental well. After incubation for various lengths of time (0.5 h, 1 h, 2 h, 4 h, and 12 h) at 37◦ C, the quantity of biofilm in each well was also measured via an XTT reduction assay. The metabolic activity of the biofilm in each experimental well was assessed and compared to the growth control for further analysis.
Statistical analysis All experiments were repeated at least two times, and the standard deviation from the mean of each tested strain was calculated. The effects of ethanol on the different phases of T. asahii biofilm were analyzed using one-way and two-way analysis of variance (ANOVA) with SPSS 16.0 (Statistical Product and Service Solutions, IBM SPSS, USA). A P50% T. asahii adhesion in all strains.
The effect of ethanol on the germination phase of T. asahii biofilm The effects of ethanol on the germination phase of T. asahii were concentration-dependent (Fig. 2). Lower concentrations (0.125% and 0.25%) of ethanol significantly induced
Inhibitory effects of ethanol on the formation and mature phases of T. asahii biofilm After T. asahii cell adhesion, the addition of ethanol suppressed the normal development of T. asahii biofilm. Ethanol demonstrated inhibitory activity against biofilm formation in concentration- and time-dependent manners when it was present at a concentration of 2% or higher, with increasing concentrations and incubation times resulting in the reduced metabolic activity of the T. asahii cells (Fig. 3). Further, exposure to 25% ethanol for 8 h or 50% ethanol for 4 h inhibited T. asahii biofilm formation by 99.15% and 99.03%, respectively. After 48 h, corresponding to the formation of the mature biofilm of T. asahii (the late phase of biofilm formation), only ethanol concentrations of 15% or higher inhibited the metabolic
Figure 1. Inhibitive effects of ethanol at the adhesion phase of T. asahii biofilm. Error bars represent SDs. ∗ means P
