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Inhibition activity of Lactobacilli supernatant against fungal-bacterial multispecies biofilms on silicone
Microbial Pathogenesis 113 (2017) 197–201
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition activity of Lactobacilli supernatant against fungal-bacterial multispecies biofilms on silicone
T
Yulong Tana,∗,1, Matthias Leonharda,1, Doris Moserb, Berit Schneider-Sticklera a b
Department of Otorhinolaryngology, Division of Phoniatrics-Logopedics, Medical University of Vienna, Vienna, Austria Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Keywords: Mixed fungal-bacterial biofilm Probiotic Silicone voice prostheses
Fungal-bacterial multispecies biofilms play a major role in failure of medical silicone devices, such as voice prostheses in laryngectomiy. In this study, we determined the effect of Lactobacilli supernatant (cell free) on mixed biofilm formation of fungi and bacteria on silicone in vitro. Lactobacilli supernatant inhibited the adhesion (90 min) of mixed fungi and bacteria species with an efficiency of > 90%. Mixed biofilm formation and the metabolic activity of the biofilms were inhibited by 72.23% and 58.36% by Lactobacilli supernatant. The examination using confocal laser scanning microscopy and scanning electron microscopy confirmed that Lactobacilli supernatant inhibited the growth of mixed biofilm and damaged the cells. Moreover, Lactobacilli supernatant also inhibited Candida yeast–to-hyphal transition. Therefore, Lactobacilli supernatant may serve as a possible antibiofilm agent to limit biofilm formation on voice prostheses.
1. Introduction Microbial biofilms are three-dimensional structured microbial communities, which are found on the surfaces of the medical devices, for example, voice prostheses [1,2]. As a standard method for voice rehabilitation after total laryngectomy, the voice prosthesis is inserted in a surgically created tracheoesophageal fistula [3]. However, the biofilm on the surface of the prosthesis increases the risk of infection and limits its life time [4]. These biofilms are well-defined as microbial multispecies communities of different bacterial and fungal species. The main fungal species identified are Candida albicans and Candida tropicalis. Several bacterial members of the skin flora of the host and commensal oral also have been detected, such as Staphylococcus, Streptococcus, Rothia dentocariosa and Candida spp. [5–8]. Inter-kingdom co-operations such as increased cell-surface adhesion and colonization and enhanced resistance to antimicrobials have been reported [9,10]. Therefore, new therapy methods for biofilms on medical devices should be investigated. Probiotics are described as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO 2001). Several food products based on probiotics have been used for nutritional or therapeutic purposes [11,12]. In recent years, the effects of probiotics on microorganisms and biofilms have been investigated, which has proven the ability of probiotics, such as
∗
1
Lactobacilli, to inhibit several bacterial or fungal pathogens growth and biofilm formation in vitro [13–15]. However, most of the work reported only focused on the effect of probiotics on single species biofilm, which is different from the multispecies biofilm. In our previous work, the mixed fungal-bacterial biofilms were grown on silicone, which is the mainly used material for voice prostheses [1]. In order to understand the mechanism of the antibiofilm effect of exometabolites produced by probiotic Lactobacillus and explore the application of the secondary metabolites in supernatants for biofilm treatment in future, in this study, the activities of Lactobacilli supernatant (cell free) on the mixed biofilm biomass and cell viability were investigated. Scanning electron microscopy and confocal laser scanning microscopy were used to determine the biofilm structure and live/dead cells of biofilm. 2. Materials and methods 2.1. Bacterial strains The fungal and bacterial pathogens used in this study were Candida albicans, Candida tropicalis, Streptococcus salivarius, R. dentocariosa, Staphylococcus epidermidis, which were clinical isolated by the Department of Otorhinolaryngology, Division of PhoniatricsLogopedics, Medical University of Vienna. Dysfunctional voice
Corresponding author. E-mail address: [email protected] (Y. Tan). Both authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.micpath.2017.10.051 Received 27 September 2017; Received in revised form 25 October 2017; Accepted 26 October 2017 Available online 28 October 2017 0882-4010/ © 2017 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 113 (2017) 197–201
Y. Tan et al.
2.6. Effect of supernatant on Candida yeast–to-hyphal transition
prostheses due to biofilm formation were explanted from the tracheoesophageal fistulas and processed within 24 h. The prostheses were vortexes in 5 ml PBS for 3 min, the microbial specimen was isolated and stored in −80 °C, and thawed before use. Lactobacillus rhamnosus was obtained from the Department of Microbiology, Medical University of Vienna.
Candida yeast–to-hyphal transition was carried out according to Cruz et al. [18]. Candida cells (OD600 0.1) were incubated with or without L. rhamnosus supernatant in YPD with 10% fetal bovine serum (FBS). After 4 h, hyphal morphology was observed by microscopy. 2.7. Scanning electron microscopy (SEM)
2.2. The cell free supernatant preparation
Mixed biofilms on silicone plates were fixed with 3% glutaraldehyde overnight, and then chemically-dried with hexamethyldisilazane. The platelets were sputter coated with gold and visualized with SEM (JSM 6310, Jeol Ltd, Akishima, Tokyo, Japan) at an acceleration voltage of 15 kV.
L. rhamnosus were inoculated anaerobically (Anaerobic jar, SigmaAldrich, Austria) at 37 °C for 24 h at 150 rpm in brain heart infusion broth (BHI, Sigma-Aldrich, Austria). The cell-free supernatant was centrifuged at 10000 rpm for 10 min to remove all cells and filtersterilized with 0.2 μM pore-size syringe filter (Sarstedt AG & Co, Germany).
2.8. Confocal scanning laser microscopy (CLSM) Live/dead analysis with CSLM was performed as previously described [16]. In brief, mixed biofilms on silicone with or without L. rhamnosus supernatant were formed and washed with PBS as described above, and then stained with Live/Dead® BacLight™ Bacterial Viability and Counting Kit (L34856, Invitrogen) following the manufacturer's instructions.
2.3. Growth of mixed biofilms on silicone plates Mixed biofilms on silicone platelets were performed according to our previous work [16]. In brief, 3-mm-diameter medical grade silicone platelets (Websinger, Austria) were placed into each well of 96-wells microtiter plates. Microbes (bacteria and Candida) were grown overnight and diluted with BHI media to OD600 = 0.01. The fungal and bacterial suspensions were mixed equally. 200 μl mixed suspension was added to each well. Non-adherent cells were removed by washing the wells with PBS after 90-min-adhesion phase. The microtiter plates were again incubated at 37 °C for 48 h.
2.9. Statistical analysis Data were expressed as mean values ± standard deviations (SD) of triplicate from three independent experiments. Statistical analysis was performed by t-test and a value of p < 0.05 was considered statistically significant.
2.4. Antiadhesion activity of supernatant 3. Results
Mixed cell suspensions were prepared as described above. 150 μl mixed suspension was added to each well containing silicone platelet together with 100 μl L. rhamnosus supernatant. In control group, equal volumes of mixed suspension and fresh BHI media were added. After 90 min, the silicone platelets were washed with PBS. The adhesion of cells on the silicone was evaluated by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD, USA), which is based on bioreduction of 2-(2- methoxy- 4- nitrophenyl)- 3- (4- nitrophenyl)- 5- (2,4- disulfophenyl)- 2H-tetrazolium (WST-8). The amount of water-soluble formazan dye produced is proportional to the number of microbes adhered to the silicone surface [17].
3.1. Antiadhesion of Lactobacillus supernatant First, we determined the L. rhamnosus supernatant capable of inhibiting mixed species adhesion to silicone. It was observed that supernatant showed significant anti-adhesion activity against the mixed species (Fig. 1). 91.02% adhesion was prevented by L. rhamnosus supernatant. 3.2. Inhibitive effect of Lactobacillus supernatant on biofilm formation Lactobacillus supernatant was found to significantly reduce the biofilm formation on silicone. As shown in Fig. 2, supernatant reduced
2.5. Antibiofilm activity of supernatant Mixed cell suspensions were prepared as described above. 100 μl (high concentration, HC) or 50 μl (low concentration, LC) Lactobacillus supernatant were added to each well of microtiter plates. 150 μl mixed cell suspensions were also added. In each well the total volume was adjusted to 250 μl using BHI. In control groups, the wells were added to BHI and the mixed cell suspensions without adding Lactobacillus supernatant. The quantification of biofilm formation was performed by the crystal violet (CV) method. After incubation at 37 °C for 48 h, the microtiter plates were washed with PBS and stained with 250 μl 1% crystal violet for 15 min. The excess of stain was removed and 250 μl of 30% acetic acid was added to each well. The absorbance at 570 nm was measured. The cell viability inside the biofilm was assessed by CCK-8 method. After incubation at 37 °C for 48 h, the microtiter plates were washed with PBS. 25 μl of the CCK-8 solution was added to each well of the plate. The microtiter plates were incubated for 2 h with protection from light. The absorbance was measured at 450 nm using a microplate reader.
Fig. 1. Inhibition of cell-surface initial interaction by L. rhamnosus supernatant. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p < 0.05 for comparison between the untreated and supernatant-treated groups.
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3.4. SEM images In the presence or absence of Lactobacillus supernatant, mixed biofilms observed by SEM are shown in Fig. 4. The mixed species biofilm formed the compact and multilayer biofilm, in which bacteria adhered to the fungi. In contrast, incubation with supernatant resulted in less biofilm and more single cells on the surface of the silicone. 3.5. CLSM images CLSM images (Fig. 5) showed mixed biofilm on the silicone surface with or without Lactobacillus supernatant. In control group, the result demonstrated most colonies and microbe in green, which indicates that microorganisms were viable and formed mixed biofilm. However, in the presence of Lactobacillus supernatant, more dead bacteria and Candida (red) were seen and there were almost no colonies.
Fig. 2. Inhibition effect of L. rhamnosus supernatant on mixed species biofilm formation. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p < 0.05 for comparison between the untreated and supernatant-treated groups.
3.6. Effect of Lactobacillus supernatant on Candida yeast–to-hyphal transition Finally, we evaluated the effect of Lactobacillus supernatant on hyphal development. As shown in Fig. 6, microscopic observation of incubation with Lactobacillus supernatant revealed marked inhibition of hyphae (Fig. 6B), whereas the control group presented an abundant component of hyphae(Fig. 6A). 4. Discussion The ability of probiotics, such as Lactobacillus species, to inhibit growth and biofilm formation by various pathogens has drawn more interest [19,20]. However, the antibiofilm effect of Lactobacillus mainly on single species pathogen and the mechanisms involved in are still not very clear. In this study, we firstly formed a mixed species biofilm of several bacteria and Candida, which are common to be detected on the surface of the voice prosthesis made of silicone. Silicone, as medical material, is widely used due to its mechanical properties and biocompatibility. However, silicone is susceptible to be colonized by microbes and a biofilm can form rapidly on the surfaces. Therefore, we evaluated the effect of Lactobacillus supernatant against mixed biofilms formation on the silicone. The results demonstrated the ability of Lactobacillus supernatant in vitro to inhibit the adhesion and the mixed biofilms formation. Biofilm formation involves several steps, such as planktonic cell adherence to the surface of materials, microbe aggregate to form colonies and produce extracellular polymeric matrix, the biofilm disperses microorganisms to the environment [16,21]. In these mixed biofilms, different species can communicate through cell-cell signaling, gene expression, drug resistance and other ways [22]. The first step of biofilm formation is the microbial adhesion. In this study, Lactobacillus supernatant reduced the adhesion stage of microorganisms, so that the further development of biofilms was blocked. The mechanisms of antiadhesion and antibiofilm by Lactobacillus
Fig. 3. Inhibition effect of L. rhamnosus supernatant on the metabolic activity of mixed species biofilm. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p < 0.05 for comparison between the untreated and supernatant-treated groups.
the mixed biofilms by 72.23% with high concentration. Incubation with low concentration supernatant of Lactobacillus also showed inhibition activity against mixed biofilm by 37.28%.
3.3. Effect of Lactobacillus supernatant on cell viability of biofilm In addition to the inhibition of Lactobacillus supernatant on mixed biofilms formation, the effects of supernatant against cell viability were also analyzed. A significant reduction in the cell viability in the biofilms compared to the group without supernatant was observed. The highest killing was 58.36% after 24 h of culture with supernatant (Fig. 3).
Fig. 4. SEM images of mixed species biofilm formations with media supplemented without (A) or with (B) supernatant.
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Fig. 5. CLSM images of mixed species biofilm formations with media supplemented without (A) or with (B) supernatant. Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the three-dimensional staining pattern for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, × 20. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Effect of L. rhamnosus supernatant on hyphal growth of C. albicans. C. albicans was grown in YPD medium containing 10% FBS in the absence (A) or presence (B) of supernatant. Samples were withdrawn after incubation at 37 °C for 4 h, and photographed.
Funding
supernatant are still not very clear. Hence, we further evaluated the effect of Lactobacillus supernatant on cell viability of biofilm. The result demonstrated the significant reduction in the cell viability of the test biofilm. The images of SEM and CLSM also confirmed this result, which indicated that Lactobacillus secreted exometabolites with antimicrobial activity in the supernatants, such as low molecular compounds, hydrogen peroxide and proteinaceous elements [23–26]. As one of the most important exometabolites of Lactobacillus, biosurfactants are able to disrupt the physical membrane structure or protein conformations, which results in cell lysis and metabolite leakage [27,28]. Moreover, biosurfactants have the ability to changes material surface energy, such as silicone, thus to thwart the microbial adhesion onto material surfaces [29,30]. The hypha formation, play an important role in the candidiasis pathogenesis, is an essential step in Candida biofilm maturation [31,32]. The hyphae formation can enhance penetration of Candida into the material that cells adhere to, thus to compromise the function of material [33]. Furthermore, bacteria adhere to Candida hyphae to form the dense mixed biofilm in mixed Candida and bacteria species, which increased colonization and enhanced drug resistance [34]. The results in this work showed that Lactobacillus supernatant can suppress C. albicans yeast-to-hyphal transition, which might ascribe to the hyphagrowth gene expression modulation by Lactobacilli [35,36]. In conclusion, the results in the present study exhibited the effect of Lactobacillus supernatant against mixed Candida/bacteria adhesion, biofilms formation and cell viability of biofilm on silicone. The mechanisms could be attributed to the exometabolites in the supernatant that disrupt the cell structure, suppress the hyphae formation and interfere with the interaction between the cells and material. Although the studies about the mechanisms and in-vivo work are needed to investigate further, this work still promises probiotics to be used for biofilm control.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interest The authors claim no conflict of interest. Submission declaration The work described above has not been published previously or under consideration for publication elsewhere. References [1] Y. Tan, M. Leonhard, D. Moser, S. Ma, B. Schneider-Stickler, Long-term antibiofilm activity of carboxymethyl chitosan on mixed biofilm on silicone, Laryngoscope 126 (12) (2016) E404–E408. [2] M. Leonhard, B. Schneider-Stickler, Voice prostheses, microbial colonization and biofilm formation, Adv. Exp. Med. Biol. 830 (2015) 123–136. [3] A.Y. Attieh, J. Searl, N.H. Shahaltough, M.M. Wreikat, D.S. Lundy, Voice restoration following total laryngectomy by tracheoesophageal prosthesis: effect on patients' quality of life and voice handicap in Jordan, Health. Qual. Life Out. 6 (2008) 26. [4] J.J. Oosterhof, K.J. Buijssen, H.J. Busscher, B.F. van der Laan, H.C. van der Mei, Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses, Appl. Environ. Microbiol. 72 (5) (2006) 3673–3677. [5] P.D. Marsh, Microbiology of dental plaque biofilms and their role in oral health and caries, Dent. Clin. North Am. 54 (3) (2010) 441–454. [6] L.O. Sanchez-Vargas, D. Estrada-Barraza, A.J. Pozos-Guillen, R. Rivas-Caceres, Biofilm formation by oral clinical isolates of Candida species, Arch. Oral Biol. 58 (10) (2013) 1318–1326. [7] C. Vuotto, F. Longo, G. Donelli, Probiotics to counteract biofilm-associated infections: promising and conflicting data, Int. J. Oral Sci. 6 (4) (2014) 189–194. [8] C.C. Wu, C.T. Lin, C.Y. Wu, W.S. Peng, M.J. Lee, Y.C. Tsai, Inhibitory effect of Lactobacillus salivarius on Streptococcus mutans biofilm formation, Mol. Oral Microbiol. 30 (1) (2015) 16–26. [9] D. Montelongo-Jauregui, A. Srinivasan, A.K. Ramasubramanian, J.L. Lopez-Ribot, An in vitro model for oral mixed biofilms of Candida albicans and Streptococcus gordonii in synthetic saliva, Front. Microbiol. 7 (2016) 686.
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition activity of Lactobacilli supernatant against fungal-bacterial multispecies biofilms on silicone
T
Yulong Tana,∗,1, Matthias Leonharda,1, Doris Moserb, Berit Schneider-Sticklera a b
Department of Otorhinolaryngology, Division of Phoniatrics-Logopedics, Medical University of Vienna, Vienna, Austria Department of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna, Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Keywords: Mixed fungal-bacterial biofilm Probiotic Silicone voice prostheses
Fungal-bacterial multispecies biofilms play a major role in failure of medical silicone devices, such as voice prostheses in laryngectomiy. In this study, we determined the effect of Lactobacilli supernatant (cell free) on mixed biofilm formation of fungi and bacteria on silicone in vitro. Lactobacilli supernatant inhibited the adhesion (90 min) of mixed fungi and bacteria species with an efficiency of > 90%. Mixed biofilm formation and the metabolic activity of the biofilms were inhibited by 72.23% and 58.36% by Lactobacilli supernatant. The examination using confocal laser scanning microscopy and scanning electron microscopy confirmed that Lactobacilli supernatant inhibited the growth of mixed biofilm and damaged the cells. Moreover, Lactobacilli supernatant also inhibited Candida yeast–to-hyphal transition. Therefore, Lactobacilli supernatant may serve as a possible antibiofilm agent to limit biofilm formation on voice prostheses.
1. Introduction Microbial biofilms are three-dimensional structured microbial communities, which are found on the surfaces of the medical devices, for example, voice prostheses [1,2]. As a standard method for voice rehabilitation after total laryngectomy, the voice prosthesis is inserted in a surgically created tracheoesophageal fistula [3]. However, the biofilm on the surface of the prosthesis increases the risk of infection and limits its life time [4]. These biofilms are well-defined as microbial multispecies communities of different bacterial and fungal species. The main fungal species identified are Candida albicans and Candida tropicalis. Several bacterial members of the skin flora of the host and commensal oral also have been detected, such as Staphylococcus, Streptococcus, Rothia dentocariosa and Candida spp. [5–8]. Inter-kingdom co-operations such as increased cell-surface adhesion and colonization and enhanced resistance to antimicrobials have been reported [9,10]. Therefore, new therapy methods for biofilms on medical devices should be investigated. Probiotics are described as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO 2001). Several food products based on probiotics have been used for nutritional or therapeutic purposes [11,12]. In recent years, the effects of probiotics on microorganisms and biofilms have been investigated, which has proven the ability of probiotics, such as
∗
1
Lactobacilli, to inhibit several bacterial or fungal pathogens growth and biofilm formation in vitro [13–15]. However, most of the work reported only focused on the effect of probiotics on single species biofilm, which is different from the multispecies biofilm. In our previous work, the mixed fungal-bacterial biofilms were grown on silicone, which is the mainly used material for voice prostheses [1]. In order to understand the mechanism of the antibiofilm effect of exometabolites produced by probiotic Lactobacillus and explore the application of the secondary metabolites in supernatants for biofilm treatment in future, in this study, the activities of Lactobacilli supernatant (cell free) on the mixed biofilm biomass and cell viability were investigated. Scanning electron microscopy and confocal laser scanning microscopy were used to determine the biofilm structure and live/dead cells of biofilm. 2. Materials and methods 2.1. Bacterial strains The fungal and bacterial pathogens used in this study were Candida albicans, Candida tropicalis, Streptococcus salivarius, R. dentocariosa, Staphylococcus epidermidis, which were clinical isolated by the Department of Otorhinolaryngology, Division of PhoniatricsLogopedics, Medical University of Vienna. Dysfunctional voice
Corresponding author. E-mail address: [email protected] (Y. Tan). Both authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.micpath.2017.10.051 Received 27 September 2017; Received in revised form 25 October 2017; Accepted 26 October 2017 Available online 28 October 2017 0882-4010/ © 2017 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 113 (2017) 197–201
Y. Tan et al.
2.6. Effect of supernatant on Candida yeast–to-hyphal transition
prostheses due to biofilm formation were explanted from the tracheoesophageal fistulas and processed within 24 h. The prostheses were vortexes in 5 ml PBS for 3 min, the microbial specimen was isolated and stored in −80 °C, and thawed before use. Lactobacillus rhamnosus was obtained from the Department of Microbiology, Medical University of Vienna.
Candida yeast–to-hyphal transition was carried out according to Cruz et al. [18]. Candida cells (OD600 0.1) were incubated with or without L. rhamnosus supernatant in YPD with 10% fetal bovine serum (FBS). After 4 h, hyphal morphology was observed by microscopy. 2.7. Scanning electron microscopy (SEM)
2.2. The cell free supernatant preparation
Mixed biofilms on silicone plates were fixed with 3% glutaraldehyde overnight, and then chemically-dried with hexamethyldisilazane. The platelets were sputter coated with gold and visualized with SEM (JSM 6310, Jeol Ltd, Akishima, Tokyo, Japan) at an acceleration voltage of 15 kV.
L. rhamnosus were inoculated anaerobically (Anaerobic jar, SigmaAldrich, Austria) at 37 °C for 24 h at 150 rpm in brain heart infusion broth (BHI, Sigma-Aldrich, Austria). The cell-free supernatant was centrifuged at 10000 rpm for 10 min to remove all cells and filtersterilized with 0.2 μM pore-size syringe filter (Sarstedt AG & Co, Germany).
2.8. Confocal scanning laser microscopy (CLSM) Live/dead analysis with CSLM was performed as previously described [16]. In brief, mixed biofilms on silicone with or without L. rhamnosus supernatant were formed and washed with PBS as described above, and then stained with Live/Dead® BacLight™ Bacterial Viability and Counting Kit (L34856, Invitrogen) following the manufacturer's instructions.
2.3. Growth of mixed biofilms on silicone plates Mixed biofilms on silicone platelets were performed according to our previous work [16]. In brief, 3-mm-diameter medical grade silicone platelets (Websinger, Austria) were placed into each well of 96-wells microtiter plates. Microbes (bacteria and Candida) were grown overnight and diluted with BHI media to OD600 = 0.01. The fungal and bacterial suspensions were mixed equally. 200 μl mixed suspension was added to each well. Non-adherent cells were removed by washing the wells with PBS after 90-min-adhesion phase. The microtiter plates were again incubated at 37 °C for 48 h.
2.9. Statistical analysis Data were expressed as mean values ± standard deviations (SD) of triplicate from three independent experiments. Statistical analysis was performed by t-test and a value of p < 0.05 was considered statistically significant.
2.4. Antiadhesion activity of supernatant 3. Results
Mixed cell suspensions were prepared as described above. 150 μl mixed suspension was added to each well containing silicone platelet together with 100 μl L. rhamnosus supernatant. In control group, equal volumes of mixed suspension and fresh BHI media were added. After 90 min, the silicone platelets were washed with PBS. The adhesion of cells on the silicone was evaluated by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD, USA), which is based on bioreduction of 2-(2- methoxy- 4- nitrophenyl)- 3- (4- nitrophenyl)- 5- (2,4- disulfophenyl)- 2H-tetrazolium (WST-8). The amount of water-soluble formazan dye produced is proportional to the number of microbes adhered to the silicone surface [17].
3.1. Antiadhesion of Lactobacillus supernatant First, we determined the L. rhamnosus supernatant capable of inhibiting mixed species adhesion to silicone. It was observed that supernatant showed significant anti-adhesion activity against the mixed species (Fig. 1). 91.02% adhesion was prevented by L. rhamnosus supernatant. 3.2. Inhibitive effect of Lactobacillus supernatant on biofilm formation Lactobacillus supernatant was found to significantly reduce the biofilm formation on silicone. As shown in Fig. 2, supernatant reduced
2.5. Antibiofilm activity of supernatant Mixed cell suspensions were prepared as described above. 100 μl (high concentration, HC) or 50 μl (low concentration, LC) Lactobacillus supernatant were added to each well of microtiter plates. 150 μl mixed cell suspensions were also added. In each well the total volume was adjusted to 250 μl using BHI. In control groups, the wells were added to BHI and the mixed cell suspensions without adding Lactobacillus supernatant. The quantification of biofilm formation was performed by the crystal violet (CV) method. After incubation at 37 °C for 48 h, the microtiter plates were washed with PBS and stained with 250 μl 1% crystal violet for 15 min. The excess of stain was removed and 250 μl of 30% acetic acid was added to each well. The absorbance at 570 nm was measured. The cell viability inside the biofilm was assessed by CCK-8 method. After incubation at 37 °C for 48 h, the microtiter plates were washed with PBS. 25 μl of the CCK-8 solution was added to each well of the plate. The microtiter plates were incubated for 2 h with protection from light. The absorbance was measured at 450 nm using a microplate reader.
Fig. 1. Inhibition of cell-surface initial interaction by L. rhamnosus supernatant. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p < 0.05 for comparison between the untreated and supernatant-treated groups.
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3.4. SEM images In the presence or absence of Lactobacillus supernatant, mixed biofilms observed by SEM are shown in Fig. 4. The mixed species biofilm formed the compact and multilayer biofilm, in which bacteria adhered to the fungi. In contrast, incubation with supernatant resulted in less biofilm and more single cells on the surface of the silicone. 3.5. CLSM images CLSM images (Fig. 5) showed mixed biofilm on the silicone surface with or without Lactobacillus supernatant. In control group, the result demonstrated most colonies and microbe in green, which indicates that microorganisms were viable and formed mixed biofilm. However, in the presence of Lactobacillus supernatant, more dead bacteria and Candida (red) were seen and there were almost no colonies.
Fig. 2. Inhibition effect of L. rhamnosus supernatant on mixed species biofilm formation. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p < 0.05 for comparison between the untreated and supernatant-treated groups.
3.6. Effect of Lactobacillus supernatant on Candida yeast–to-hyphal transition Finally, we evaluated the effect of Lactobacillus supernatant on hyphal development. As shown in Fig. 6, microscopic observation of incubation with Lactobacillus supernatant revealed marked inhibition of hyphae (Fig. 6B), whereas the control group presented an abundant component of hyphae(Fig. 6A). 4. Discussion The ability of probiotics, such as Lactobacillus species, to inhibit growth and biofilm formation by various pathogens has drawn more interest [19,20]. However, the antibiofilm effect of Lactobacillus mainly on single species pathogen and the mechanisms involved in are still not very clear. In this study, we firstly formed a mixed species biofilm of several bacteria and Candida, which are common to be detected on the surface of the voice prosthesis made of silicone. Silicone, as medical material, is widely used due to its mechanical properties and biocompatibility. However, silicone is susceptible to be colonized by microbes and a biofilm can form rapidly on the surfaces. Therefore, we evaluated the effect of Lactobacillus supernatant against mixed biofilms formation on the silicone. The results demonstrated the ability of Lactobacillus supernatant in vitro to inhibit the adhesion and the mixed biofilms formation. Biofilm formation involves several steps, such as planktonic cell adherence to the surface of materials, microbe aggregate to form colonies and produce extracellular polymeric matrix, the biofilm disperses microorganisms to the environment [16,21]. In these mixed biofilms, different species can communicate through cell-cell signaling, gene expression, drug resistance and other ways [22]. The first step of biofilm formation is the microbial adhesion. In this study, Lactobacillus supernatant reduced the adhesion stage of microorganisms, so that the further development of biofilms was blocked. The mechanisms of antiadhesion and antibiofilm by Lactobacillus
Fig. 3. Inhibition effect of L. rhamnosus supernatant on the metabolic activity of mixed species biofilm. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p < 0.05 for comparison between the untreated and supernatant-treated groups.
the mixed biofilms by 72.23% with high concentration. Incubation with low concentration supernatant of Lactobacillus also showed inhibition activity against mixed biofilm by 37.28%.
3.3. Effect of Lactobacillus supernatant on cell viability of biofilm In addition to the inhibition of Lactobacillus supernatant on mixed biofilms formation, the effects of supernatant against cell viability were also analyzed. A significant reduction in the cell viability in the biofilms compared to the group without supernatant was observed. The highest killing was 58.36% after 24 h of culture with supernatant (Fig. 3).
Fig. 4. SEM images of mixed species biofilm formations with media supplemented without (A) or with (B) supernatant.
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Fig. 5. CLSM images of mixed species biofilm formations with media supplemented without (A) or with (B) supernatant. Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the three-dimensional staining pattern for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, × 20. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Effect of L. rhamnosus supernatant on hyphal growth of C. albicans. C. albicans was grown in YPD medium containing 10% FBS in the absence (A) or presence (B) of supernatant. Samples were withdrawn after incubation at 37 °C for 4 h, and photographed.
Funding
supernatant are still not very clear. Hence, we further evaluated the effect of Lactobacillus supernatant on cell viability of biofilm. The result demonstrated the significant reduction in the cell viability of the test biofilm. The images of SEM and CLSM also confirmed this result, which indicated that Lactobacillus secreted exometabolites with antimicrobial activity in the supernatants, such as low molecular compounds, hydrogen peroxide and proteinaceous elements [23–26]. As one of the most important exometabolites of Lactobacillus, biosurfactants are able to disrupt the physical membrane structure or protein conformations, which results in cell lysis and metabolite leakage [27,28]. Moreover, biosurfactants have the ability to changes material surface energy, such as silicone, thus to thwart the microbial adhesion onto material surfaces [29,30]. The hypha formation, play an important role in the candidiasis pathogenesis, is an essential step in Candida biofilm maturation [31,32]. The hyphae formation can enhance penetration of Candida into the material that cells adhere to, thus to compromise the function of material [33]. Furthermore, bacteria adhere to Candida hyphae to form the dense mixed biofilm in mixed Candida and bacteria species, which increased colonization and enhanced drug resistance [34]. The results in this work showed that Lactobacillus supernatant can suppress C. albicans yeast-to-hyphal transition, which might ascribe to the hyphagrowth gene expression modulation by Lactobacilli [35,36]. In conclusion, the results in the present study exhibited the effect of Lactobacillus supernatant against mixed Candida/bacteria adhesion, biofilms formation and cell viability of biofilm on silicone. The mechanisms could be attributed to the exometabolites in the supernatant that disrupt the cell structure, suppress the hyphae formation and interfere with the interaction between the cells and material. Although the studies about the mechanisms and in-vivo work are needed to investigate further, this work still promises probiotics to be used for biofilm control.
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