Inhibitory effect of lactobacilli supernatants on biofilm and filamentation of C. 1 albicans

9 Probiotic Lactobacillus strains had been investigated for the potential to protect against 10 infection caused by the major fungal pathogen of human, Candida albicans . Besides 11 antifungal activity, lactobacilli demonstrated a promising inhibitory effect on biofilm 12 formation and filamentation of C. albicans . On the other hand, two commonly isolated 13 non-albicans Candida species, C. tropicalis and C. parapsilosis , have similar 14 characteristics in filamentation and biofilm formation with C. albicans . However, there is 15 scant information of the effect of lactobacilli on the two species. In this study, biofilm 16 inhibitory effects of L. rhamnosus ATCC 53103, L. plantarum ATCC 8014, and L. 17 acidophilus ATCC 4356 were tested on the reference strain C. albicans SC5314 and six 18 bloodstream isolated clinical strains, two each of C. albicans , C. tropicalis , and C. 19 parapsilosis . Cell-free culture supernatants (CFSs) of L. rhamnosus and L. plantarum 20 significantly inhibited in vitro biofilm growth of C. albicans and C. tropicalis . L. 21 acidophilus , conversely, had little effect on C. albicans and C. tropicalis but was more 22 effective on inhibiting C. parapsilosis biofilms. Neutralized L. rhamnosus CFS at pH 7 23 retained the inhibitory effect, suggesting that exometabolites other than lactic acid 24 produced by the Lactobacillus strain might be accounted for the effect. Furthermore, we 25 evaluated the inhibitory effects of L. rhamnosus and L. plantarum CFSs on the


Introduction
Candida species are commensal fungi that colonize healthy human skin, gastrointestinal and genital tracts.They are also opportunistic pathogens, leading to mucosal and systemic infections (Kim and Sudbery, 2011).Candida are one of the most important agents of fungal infection worldwide and the most frequently isolated fungal species in healthcare-associated infections (Brown et al., 2012).Over 90% of invasive Candida infections were caused by five species, Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei, with C. albicans accounted for 47-66% of cases (Pfaller and Diekema, 2007;Pfaller et al., 2019).Being the predominant cause of fungal infection, C. albicans has become the best-studied Candida species (Kabir et al., 2012).Although C. albicans remains as the most common isolated single species, a progressive shift to increased prevalence of non-albicans Candida species has been observed over the past decades (Pfaller et al., 2019).In the global SENTRY antifungal surveillance program, the frequency of C. albicans was reported to gradually decrease from 57.4% of invasive Candida isolates in 1997-2001to 46.4% in 2015-2016(Pfaller et al., 2019)).
Among the four common isolated non-albicans species, C. tropicalis and C. parapsilosis are phylogenetically closely related to C. albicans (Butler et al., 2009).Similar to C. albicans, C. tropicalis and C. parapsilosis can undergo yeast-filament transition, which is an important virulence factor of C. albicans for invading host epithelial cells and causing tissue damage (Sudbery, 2011).C. albicans and C. tropicalis produce true hyphae and pseudohyphae while C. parapsilosis produces only pseudohyphae (Lackey et al., 2013).
These three species are also capable of forming biofilm composed of yeast cells and filaments.
Candida biofilm formed in vivo on abiotic surfaces of indwelling medical devices, such as dentures, endotracheal tubes, prosthetic device, and various types of catheters, can lead to device failure and infections, including fatal catheter-related bloodstream infections (Ramage et al., 2006).Moreover, Candida biofilm has shown significantly decreased susceptibility to some commonly used antifungal agents, such as fluconazole and amphotericin B, when compared to the planktonic cells (Ramage et al., 2001;Pierce et al., 2008).Biofilm protects Candida cells from antifungal agents, leading to persistent infection that is difficult to eliminate without removal of the infected implant (Ramage et al., 2006).
In view of the emergence of antifungal resistance, the use of probiotic Lactobacillus species has been suggested as an alternative therapeutic option against Candida infection (Matsubara et al., 2016a).Several clinical trials reported that the administration of Lactobacillus improve outcome of vaginal candidiasis and reduce colonization of Candida in oral cavity of elderly people and in gastrointestinal tract of preterm neonates (Roy et al., 2014;Kovachev and Vatcheva-Dobrevska, 2015;Kraft-Bodi et al

2015).
In vitro studies also demonstrated that lactobacilli possess anticandidal ability, inhibitory effect on adhesion to host cells, and inhibitory effect on biofilm formation and filamentation of C. albicans (Strus et al., 2005;Köhler et al., 2012;Coman et al., 2015;Vilela et al., 2015;Matsubara et al., 2016b;Song and Lee, 2017).Interestingly, as demonstrated with cell-free supernatants and in agar overlay assays, direct interaction of lactobacilli with C. albicans cells is unnecessary for the inhibitory effect, suggesting that the effect is exhibited by the metabolites produced by lactobacilli (Matsubara et al., 2016b;Liao et al., 2019).
Although the effect of lactobacilli on C. albicans has been extensively studied, there is a relative lack of studies on non-albicans species.The aim of this study was to investigate the inhibitory effect of L. rhamnosus, L. plantarum, and L. acidophilus on the biofilm formation of C. albicans and closely related non-albicans species, C. tropicalis and C.
parapsilosis.In addition, we evaluated the change in filamentation and the expression of filamentation-and biofilm-related genes of C. albicans and C. tropicalis when incubated with cell-free supernatants of L. rhamnosus and L. plantarum.

2
Materials and Methods for 18 h, the liquid cultures were centrifuged to separate spent media and bacterial cells.

Strains and Culture Conditions
Cell-free supernatant (CFS) was prepared by filtering the spent media through a membrane filter with a 0.22 μm pore size (Merck Millipore, USA).MColorpHast pH indicator strip (Millipore, USA) was used to measure the pH of CFS.The harvested Lactobacillus cells were washed twice with PBS and resuspended in MRS broth to 1 × 10 7 CFU/ml.
The incubation of biofilm was divided into two parts, adhesion phase and formation phase (Kuhn et al., 2002).Briefly, in adhesion phase, 100 μl of Candida inoculum were seeded into the wells of flat bottom 96-well plates.Plates were prepared in duplicate and then incubated statically at 37 °C for 90 mins.After incubation, spent medium was aspirated from the well.The wells were washed with PBS to remove non-adherent cells.
In the formation phase, 100 μl of RPMI medium and 50 μl of Lactobacillus CFS, living cell suspension, or MRS broth as control were added per wells.There were five replicate wells for each condition per plate.The plates were incubated statically at 37 °C for an additional 24 h.After incubation, a MColorpHast pH strip was used to check the pH of spent medium.The biofilms were quantified by two methods, XTT reduction assay and crystal violet staining.
To examine whether a lower pH contributed to the inhibitory effect of CFS, the assay was repeated using MRS broth acidified to pH 4.0 with lactic acid, and LGG CFS neutralized to pH 7.0 with sodium hydroxide.

Filamentation inhibition
The C. albicans and C. tropicalis strains were tested against the CFSs of lactobacilli LGG or LP8014, which were prepared from 18h-old culture as previously described and stored at -20°C until use.MRS broth was used as control.The assay was performed following the method described by Wang et al. with some modifications (Wang et al., 2017).In a 1.5 ml Eppendorf tube, 144 μl of Candida suspension in RPMI medium at concentration of 1 × 10 7 CFU/ml were mixed with 300 μl of Lactobacillus CFS or MRS broth and then topped up to a total volume of 900 μl with RPMI.Additional 10% v/v of fetal bovine serum were added for C. tropicalis strains to induce filamentation.After incubating statically at 37 °C for 4 h, the mixture was vortexed for 20 s and 20 μl of it were loaded into a Fuchs-Rosenthal counting chamber (Marienfeld, Germany).Microphotographs of ten 0.0625 mm 2 squares of the chamber were captured using a AmScope MD500 digital eyepiece camera (AmScope, USA) under a light microscope with a 10× objective lens.
The numbers of filaments within the ten 0.0625 mm 2 squares were counted from the photographic images.

Gene expression in biofilm
To determine the effects of Lactobacillus CFS on the transcription of biofilm-related genes in C. albicans and C. tropicalis, the gene expression levels of ACT1, ALS1, ALS3, BCR1, EFG1, TEC1, RIP1, and UME6 of C. albicans and the corresponding orthologs in C. tropicalis were evaluated using quantitative real-time polymerase chain reaction(qPCR).Specific primer pairs were designed using the NCBI/Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)except RIP1, primers for which were adopted from previous literature (Nailis et al., 2006).The sequences of primers were listed in Table S1 and S2.
To extract RNA, biofilms of C. albicans and C. tropicalis were formed in a flat bottom, cell culture-treated 48-well plate (Thermo Fisher Scientific, USA) using the two-phase procedures described for biofilm inhibition, with the volumes of inoculum and CFS (or MRS broth) increased to 200 μl and 100 μl respectively.After incubated statically at 37 °C for 24 h, RNA was extracted from biofilms using the TRIzol™ Plus RNA Purification Kit (Thermo Fisher Scientific, USA) with homogenization done on a Fastprep-24 5G instrument (MP Biomedicals, USA) with lysing matrix Y 2ml tubes at a speed of 6.0 m/s for 30 s twice, with a cooling interval on ice for 3 minutes.The RNA was eluted with 30 μl of RNase-free water preheated to 60 °C.RNA samples were DNase treated using the TURBO DNA-free Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instruction.Complementary DNA (cDNA) was reverse transcribed from 500 ng of RNA by the High-Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Thermo Fisher Scientific, USA).cDNA samples were stored at -80 °C until use.
qPCR was performed in 10 μl reaction volume using the SsoAdvanced Universal SYBR Green Supermix (Bio-rad, USA) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA).Candida strain, as well as no template controls, was performed within a single qPCR run (96-well plate) with three technical replicates per condition.
The reactions started from a primary denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.Specificity of amplification was confirmed by melting curve analysis.Expression levels of the genes of interest were normalized to the reference genes ACT1 and RIP1 using the following formula: , where E represents the amplification efficiency and ΔCt is the difference in cycle threshold (Ct) values of the treatment and control group (Nailis et al., 2006;Hellemans et al., 2007).

Statistical Analysis
Experiments were repeated in triplicate at different occasions.Results of experiment groups in the biofilm inhibition assay were expressed as a percentage relative to the control (MRS) and compared to the control using Student's t test.Results of filamentation assay were compared to control using one-way analysis of variance (ANOVA) followed by Dunnett's test.Expression levels of genes in biofilm were expressed as NRQ as described.Student's t test was used to compare the binary logarithms of the NRQs of experimental group to control group of the same Candida strain.A p-value ≤ 0.05 was considered significant in all tests.Statistical analyses were performed using Prism (Version 9, GraphPad Software, USA and Microsoft Excel (Office 365, Microsoft, USA).

Biofilm inhibition
The LGG CFS significantly reduced the biomass of the biofilms produced by five Candida strains and the metabolic activity of three strains (Figure 1B).The inhibition was most pronounced (>50% in biomass, P < 0.01) in C. albicans, followed by C.
tropicalis.The LP8014 CFS also shown inhibitory effects to some extent and significantly reduced the biomass and metabolic activity of biofilm formed by C. albicans SC5314 (Figure 1D, P < 0.05).However, living LGG and LP8014 cell suspensions had little to no effect (Figure 1A and C).On the other hand, LA4356's cells significantly reduced both biomass and metabolic activity of the two C. parapsilosis strains whiles its CFS also had similar effect (Figure 1E and F).
Both LGG CFS and LP8014 CFS had a median pH of 4.0, but LA4356 CFS was less acidic (median pH 5).When used in inhibitory assay, the CFSs lowered the median pH of the spent medium after 24-hour incubation from 7.5 (control, MRS broth) to 6.0 (LGG), 6.5 (LP8014), and 7.0 (LA4356) respectively.The medium pH of spent medium was 7.0 in the living cell groups of the three Lactobacillus strains.However, when incubated with RPMI medium without Candida inoculum, all three Lactobacillus strains were able to reduce the medium pH to 4.5 after 24 h.
To examine whether a lower pH contributed to the inhibitory effect of CFS, the assay was

Filamentation inhibition
As the CFSs of LGG and LP8014 showed pronounced inhibitory effects on biofilms of C. LP8014 CFS when compared to control (Figure 4B).The filamentation of C. albicans A69 when incubated with CFSs was inhibited but not significantly different from the control (Figure 4C).

Gene expression in biofilm
Expression of ALS1, ALS3, BCR1, EFG1, TEC1, and UME6 genes in biofilms of C. albicans and C. tropicalis strains incubated with LGG or LP8014 CFSs were quantified by qPCR.Although being significant only in some of the strains, there were consistent downregulations in ALS1, EFG1 and TEC1 genes caused by LGG CFS, and in ALS3 and EFG1 genes by LP8014 CFS in the biofilm of the C. albicans strains SC5314, A14, and A69 (Figure 8).On the other hand, ALS3 and UME6 genes were downregulated and TEC1 gene was upregulated by both CFSs in C. tropicalis T18S and T38R.Interestingly, the expression levels of ALS1 in T38R were significantly increased to 1.648-and 1.770fold by both CFSs (Table 1).The expression level of UME6 gene was downregulated by LP8014 CFS in SC5314, T18S, and T38R and by LGG CFS in T18S, but the differences were not statistically significant.

Discussion
Lactobacillus species have been reported to exhibit an inhibitory effect on biofilm formation and filamentation of C. albicans, however few studies have described their effect on the non-albicans species.In the present study, we studied the antibiofilm effect of cell suspensions and CFSs of three parapsilosis, even when being effective on other Candida species (Parolin et al., 2015).
Our study showed an inhibitory effect of L. rhamnosus (LGG) and L. plantarum (LP8014) CFSs on Candida biofilm.This finding corroborates the results of numerous studies using other Lactobacillus species and strains (Vilela et al., 2015;James et al., 2016;Matsubara et al., 2016b;Ribeiro et al., 2017;Matsuda et al., 2018;Tan et al., 2018).The indirect interaction between Lactobacillus cells and Candida biofilm suggests that soluble exometabolites secreted into the culture medium by Lactobacillus are possibly responsible for the inhibitory effect.Production of lactic acid and the resulted low pH (4.5) environment has been accounted for candicidal activity of L. rhamnosus and Lactobacillus reuteri (Köhler et al., 2012).However, in our biofilm assay, the lowest pH of medium measured was 6.0 and caused by the addition of LGG CFS.Moreover, acidified MRS broth showed little inhibitory effect on biofilm, and the inhibitory effect was not diminished in neutralized LGG CFS (p <0.05 when compared with MRS control, except for biomass of C. tropicalis T18S and T38R) and only reduced when compared with untreated LGG CFS in C. albicans A14 and A69.These results suggested that there are other factors contributed to the inhibitory effect.
Nguyen et al. demonstrated that sodium butyrate can significantly inhibit biofilm formation of C. albicans and C. parapsilosis, by 80% and 65% respectively, at a concentration of 10 mM (Nguyen et al., 2011).Song and Lee investigated the antifungal activity of spent culture medium of L. rhamnosus and L. casei and found that the growth of C. albicans in both yeast-and hyphal-predominant conditions were significantly inhibited with 50% v/v spent culture medium (Song and Lee, 2017).However, proteinase K-treated spent culture medium of both species lost the inhibitory effect, suggesting that the effect was due to antifungal peptides.A biosurfactant produced by a Lactobacillus brevis strain CV8LAC was shown to inhibit C. albicans biofilm formation by coincubation and precoating the substrate whiles demonstrated no inhibition on the fungal growth in planktonic and sessile form (Fracchia and Allegrone, 2010;Ceresa et al., 2015).It is possible that these metabolites and similar compounds also contribute to the inhibitory effect of CFS demonstrated in our study, but further investigation is needed to identify the exact active compounds and their mechanisms.
Filaments are key structural component of mature biofilm of C. albicans (Chandra et al., 2001;Kuhn et al., 2002).Mutants of hyphal-related genes showed defects in forming hyphae and biofilm formation as well (Ramage et al., 2002;Nobile and Mitchell, 2005).
Therefore, we investigated the effect of CFSs of LGG and LP8014 on the filamentation of the C. albicans and C. tropicalis strains.The filamentation of C. albicans and C.
tropicalis cells was greatly reduced when co-incubated with the CFSs.These results are consistent with other studies that demonstrated inhibitory effects of Lactobacillus cell suspensions and CFSs on C. albicans in filamentation assay of similar designs (Vilela et al., 2015;Ribeiro et al., 2017;Matsuda et al., 2018;Rossoni et al., 2018b) (Matsubara et al., 2016b).In a monolayer model of oral epithelial cells, preincubating the epithelial cells with LGG suspension for 12 h before C. albicans infection was shown to significantly reduce hyphal length and invasion of C. albicans (Mailänder-Sánchez et al., 2017).
Environmental pH is known to induce morphological differentiation in C. albicans.
Growth in yeast from is favored in acidic conditions, whereas neutral and alkaline conditions prompt hyphal growth (Davis, 2003).We found that the addition of LGG and LP8014 CFSs reduced the pH of the medium from 7.5 of the control group (MRS broth) to between 6 and 7.As Nadeem et al. has observed that germ tube formation in C.
albicans at pH 6.4 was moderately lower than at pH 7.4 and further reduced at pH 5.4, we could not exclude the effect of the slightly decreased pH in the inhibition of filamentation observed in our study, but other metabolites of Lactobacillus are suggested to contribute (Nadeem et al., 2013).For example, in a recent study, 1-acetyl-β-carboline (1-ABC), a small molecule isolated from the culture supernatant of Lactobacillus species, was reported to block C. albicans filamentation in a concentration-dependent manner (MacAlpine et al., 2021).It was proposed that 1-ABC inhibits C. albicans Yak1 kinase, which is necessary for upregulation of hypha-induced genes (Goyard et al., 2008).
Purified 1-ABC was also shown to inhibit filamentation of C. dubliniensis and C.
tropicalis, in which Yak1 is conserved.The authors also demonstrated that acidic pH alone was insufficient to achieve the same degree of filamentation inhibition caused by Lactobacillus culture supernatant.
To elucidate the mechanisms of the antibiofilm activity of Lactobacillus CFS, we measured the expression of six biofilm-related genes, namely ALS1, ALS3, BCR1, EFG1, TEC1, and UME6, in the biofilm produced by C. albicans or C. tropicalis co-incubated with LGG and LP8014 CFSs.ALS1 and ALS3, belonging to the agglutinin-like sequence (ALS) gene family, encode cell wall glycoproteins Als1 and Als3 respectively (Hoyer, 2001).Expression of ALS1 is detectable in both yeast or hyphae form, whiles ALS3 transcribed exclusively in germ tubes and hyphae (Green et al., 2005).In C. tropicalis, Galán-Ladero et al. (Galán-Ladero et al., 2019) reported that ALS1-, ALS2-, and ALS3like genes were more upregulated in sessile cells, suggesting their roles in biofilm formation.BCR1, encoding a transcription factor Bcr1, is upregulated in C. albicans hyphae but not required for normal hyphal development (Nobile and Mitchell, 2005).
Bcr1 is required for the full expression of several cell wall proteins, including Als1 and the hyphal-specific Als3, Hwp1, and Hyr1.EFG1 encodes the transcription factor Efg1, which is essential for yeast-hyphal transition and biofilm formation of C. albicans (Stoldt et al., 1997;Ramage et al., 2002).Efg1 is activated by upstream cAMP pathway and regulates expression of hyphal-specific genes and downstream transcription factors such as Tec1 and Eed1 (Sudbery, 2011).Mancera et al. has reported that EFG1 ortholog in C.
tropicalis has conserved function on filamentation and biofilm formation (Mancera et al., 2015).TEC1 encodes transcription factor Tec1, deletion of which results in defect of hyphal formation and biofilm formation, and suppression of expression of secreted aspartyl protease (SAP) family proteins Sap4-6 in C. albicans (Schweizer et al., 2000;Nobile and Mitchell, 2005).In C. tropicalis, deletion mutants of BCR1, BRG1, TEC1, EFG1, or NDT80 homologs produced significantly less biofilm biomass, suggesting the conserved roles of these genes in biofilm formation (Tseng et al., 2020).UME6, a common downstream target of regulators Efg1, Chp1 and Ras1, strictly or partially controls the expression of hyphae-specific genes in C. albicans (Zeidler et al., 2009).
Constitutive high-level expression of UME6 orthologs in C. tropicalis and C.
To the best of our knowledge, this is the first study to examine the expression of biofilmrelated genes in C. tropicalis biofilm under a biofilm-inhibitory condition.In our study, we observed a downregulation in EFG1 and TEC1 genes by CFS in the three C. albicans strains, which is in agreement with the findings of previous studies on C. albicans biofilm (James et al., 2016;Matsuda et al., 2018;Rossoni et al., 2018a).These studies, however, also reported significant downregulation of ALS3 and BCR1 genes in Lactobacillus-inhibited biofilm, which was not the case in our results.Interestingly, in our study, the expression of ALS1, BCR1, and TEC1 genes were upregulated in C. tropicalis T38R by LGG CFS although LGG CFS was shown to suppress biofilm formation and filamentation of T38R, suggesting a strain-dependent gene response.The upregulation of ALS1 in T38R could possibly be a compensatory upregulation, which has been demonstrated in deletion mutants of ALS2 and ALS4, and of the SAP gene family in C. albicans, for the downregulation of ALS3 (Zhao et al., 2005;Naglik et al., 2008;Liu and Filler, 2011).The expression of UME6 in C. albicans SC5314, C. tropicalis T18S and T38R was downregulated by 0.434-, 0.360-(p=0.0956),and 0.609-fold respectively, suggesting that the mechanism of action of LP8014 CFS involves the repression of UME6.We selected few target genes from the biofilm regulatory network of Candida, which involves multiple signaling pathways and more than 50 genes (Finkel and Mitchell, 2011).Other well-known biofilm-related genes, such as HWP1, which encodes a cell wall adhesin required in biofilm formation, and NRG1, a negative regulator of filamentation, were not included (Braun et al., 2001;Nobile et al., 2006) .To unravel the elusive mechanisms of the effect of Lactobacillus CFS, genomic-wide transcripting profiling by technologies such as RNA-seq is a better approach (Chong et al., 2018).

Conclusion
The present study evaluated the inhibitory effect of three lactobacilli strains, LGG, tropicalis.This study demonstrated an alternative approach to control Candida biofilm.
The active components and the potential clinical application of LGG and LP8014 CFSs remain to be investigated.
repeated with MRS broth acidified to pH 4.0, and LGG CFS neutralized to pH 7.0.As shown in Figure 2, significant inhibition by acidified MRS was only found in biomass of C. albicans A14 (35.3% of reduction) and metabolic activity of T18S (12.9%) when compared to MRS control.Neutralized LGG CFS only shown significant reduced inhibitory effect on metabolic activity of C. albicans A69 and on biomass of A14 and A69 when compared to untreated LGG CFS.
albicans and C. tropicalis, their effects on the filamentation of C. albicans and C. tropicalis were further investigated.In the filamentation assay, a large number of Candida cells developed into filaments after incubating in RPMI medium mixed with MRS broth (control).However, many Candida cells remained in yeast form in the presence of Lactobacillus CFSs. Figure 3 showed the examples of cell morphology of each tested Candida strains under different conditions.Filamentations of C. albicans SC5314, C. tropicalis T18S and T38R (Figure 4A, D and E) were statistically significantly inhibited by both LGG CFS and LP8014 CFS (P < 0.05).The number of filaments of C. albicans A14 was significantly reduced by LGG CFS (P < 0.05) but not LP8014, and LA4356, on C. albicans, C. tropicalis, and C. parapsilosis.Significant inhibitory effects were demonstrated by the CFSs of LGG and LP8014 on filamentation and biofilm formation of both C. albicans and C. tropicalis.Hypha-related gene expressions in biofilm were also changed in response to the addition of CFSs.However, C. parapsilosis biofilms were not susceptible to the inhibitory effect of these two lactobacilli strains.The pathogen-specific inhibition suggests a common mechanism of action, presumably mediated by exometabolites of the lactobacilli, on C. albicans and C.
FIGURE 1. Effects of living cell suspension and CFS of three Lactobacillus strains on biofilm formation of seven Candida strains.

FIGURE 4 .
FIGURE 4. Quantification of filament in the filamentation inhibition assays.

FIGURE 5 .
FIGURE 5. Expression levels filamentation and biofilm-related genes in biofilms of C. albicans and C. tropicalis incubated with LGG or LP8014 CFSs.
., (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made C. parapsilosis was found to be less susceptible to CFSs of LGG and LP8014.Similar Candida species-specific manner of the Lactobacillus inhibitory activity was reported in previous research.Tan et al. observed that biofilm formation of C. parapsilosis was less suppressed by the supernatant of L. gasseri and L. rhamnosus than C. tropicalis and C. krusei(Tan et al., 2018).Parolin et al. investigated the antifungal activities of CFS of Lactobacillus strains isolated from vaginal swabs on C. albicans and non-albicans species but found that no strains were effective against C. krusei and C.
(Vilela et al., 2015)Matsubara et al., 2016b;Ribeiro et al., 2017)ilosis.LGG CFS shown the strongest anti-biofilm activity against C.albicans and C. tropicalis strains, followed by LP8014 CFS.The C. parapsilosis were more susceptible to LA4356 than LGG and LP8014.Previous studies have reported the anti-biofilm activity of Lactobacillus cell suspensions on C. albicans, however, we detected only a modest effect of cell suspension on Candida biofilm in our study(Vilela et al., 2015;Matsubara et al., 2016b;Ribeiro et al., 2017).It is noteworthy that different methods of preparing the cell suspensions were used.In our study, Lactobacillus cells significant for the 24-hour L. acidophilus culture, suggesting that inhibitory activity of Lactobacillus is possibly associated with the growth stage(Vilela et al., 2015).In the present study, (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made

TableTABLE 1 . Expression levels filamentation and biofilm-related genes in biofilms of C. albicans and C. tropicalis incubated with LGG or LP8014 CFSs.
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made Expression levels are expressed as mean ± SEM of the normalized relative quantity (NRQ) obtained from triplicate experiments.NRQ of control group (MRS) is constantly 1.0 and not shown.†P-value was obtained by comparing to the control group using Student's t test.Bold values denote statistical significance at p < 0.05. *