Genomic Analysis of Limosilactobacillus fermentum ATCC 23271, a Potential Probiotic Strain with Anti-Candida Activity

Limosilactobacillus fermentum (ATCC 23271) was originally isolated from the human intestine and has displayed antimicrobial activity, primarily against Candida species. Complete genome sequencing and comparative analyses were performed to elucidate the genetic basis underlying its probiotic potential. The ATCC 23271 genome was found to contain 2,193,335 bp, with 2123 protein-coding sequences. Phylogenetic analysis revealed that the ATCC 23271 strain shares 941 gene clusters with six other probiotic strains of L. fermentum. Putative genes known to confer probiotic properties have been identified in the genome, including genes related to adhesion, tolerance to acidic pH and bile salts, tolerance to oxidative stress, and metabolism and transport of sugars and other compounds. A search for bacteriocin genes revealed a sequence 48% similar to that of enterolysin A, a protein from Enterococcus faecalis. However, in vitro assays confirmed that the strain has inhibitory activity on the growth of Candida species and also interferes with their adhesion to HeLa cells. In silico analyses demonstrated a high probability of the protein with antimicrobial activity. Our data reveal the genome features of L. fermentum ATCC 23271, which may provide insight into its future use given the functional benefits, especially against Candida infections.


Introduction
Candida species are considered commensal microorganisms in humans [1]. Nevertheless, they can cause opportunistic infections, including some that are less severe and superficially located, and others of a life-threatening systemic nature, particularly in immunocompromised patients. Localized candidiasis mainly affects the mucosa of the oral cavity and vagina [2,3].
Azoles are commonly used to treat candidiasis [4]. These drugs are fungistatic, meaning that an efficient immune system is necessary to control the infection and resolve its C. albicans CAS (vaginal isolate), C. krusei GJFD (vaginal isolate), C. parapsilosis FSG (oral isolate) and C. parapsilosis RCL (vaginal isolate). Probiotic properties were also evaluated against clinical Candida strains isolated from oral and vaginal cavities, belonging to the Culture Collection Sector of the Biology Laboratory of the Federal Institute of Education, Science and Technology of Maranhão. L. fermentum ATCC 23271 strains were cultivated in De Man, Rogosa, and Sharpe (MRS) agar and broth (Difco Laboratories, Detroit, MI, USA) under anaerobic conditions, while the other bacteria and yeasts were grown in brain heart infusion (BHI) agar/broth and Sabouraud agar, respectively (Difco Laboratories, Detroit, MI, USA), under aerobic conditions. All microorganisms were incubated at 37 • C for 24 h.

Whole-Genome Sequencing of Limosilactobacillus fermentum ATCC 23271
Genome sequencing of L. fermentum ATCC 23271 was performed by Neoprospecta using the Illumina HiSeq4000. Genomic DNA was extracted using the PureLink ® Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The complete genome was sequenced using the Illumina MiSeq paired library approach and prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). The sequence readings were assembled using A5 software [38] and processed for adapter cutting, quality filtering and error correction to generate the contigs and scaffolds. In addition, CAP3 software [39] was used to improve the scaffolding assembly, cut low-quality regions and correct erroneous links between contigs. The pre-assembled genomic DNA sequences were annotated using the software tool Prokka [40].

Genome Analysis
The annotated sequences of the L. fermentum ATCC 23271 genome were analyzed using Rapid Annotation using Subsystem Technology (RAST) (available at https://rast. nmpdr.org/rast.cgi accessed on 10 January 2017). The Basic Local Alignment Search Tool (BLAST) was used to search for similarities in the protein databases of the National Center for Biotechnology Information (NCBI) and Universal Protein Resource. Phaster (https://phaster.ca/ accessed on 17 May 2021) was used to search for prophage-like clusters. Orthovenn (http://probes.pw.usda.gov/OrthoVenn accessed on 17 may 2021) was used to search for orthologous proteins common to the lineages L. fermentum 2760, L. fermentum DR 9, L. fermentum 3872, L. fermentum CECT 5716 and L. fermentum AGR 1485 [41]. To search for genes associated with bacteriocins, the BAGEL4 program (available at http://bagel4.molgenrug.nl/ accessed on 23 June 2020) was used, and a protein with similarity to another known bacteriocin was first analyzed using the Signal P 5.0 Server to identify the signal peptide cleavage region. The mature protein sequence was analyzed in the CAMP database using four machine learning algorithms, including Support Vector Machine, Random Forest, Artificial Neural Network and Discriminant Analysis, to predict its antimicrobial activity [42]. A phylogenetic tree was generated using the nucleotide sequence using the TYGS server (http://tygs.dsmz.de accessed on 3 February 2021) [43] using genomic sequences deposited in GenBank. To analyze evolutionary relationships between species, Mauve linked in Geneious software [44] was used. PathogenFinder v. 1.1 [45] was used to estimate the pathogenicity of the strain in human hosts. ResFinder v. 3.1 [46] and the Comprehensive Antibiotic Resistance Database (CARD) v. RGI 5.1.0, CARD 3.0.7 [47] were used to search for antimicrobial resistance genes. Virulence determinants were analyzed using VirulenceFinder v. 2.0 [48]. The rapid identification and annotation of prophage sequences within the genome of L. fermentum ATCC 23271 were performed using the phage search tool enhanced release (PHASTER) [49]. The CRISPR Finder tool was used to detect CRISPR direct repeats and spacers [50].

Tolerance to Gastrointestinal Conditions
The tolerance of L. fermentum ATCC 23271 to acidic pH (pH = 2 and 4) and bile salts (0.5% and 1%, Oxgall, Sigma-Aldrich) was verified as described by Monteiro et al. [52]. Initially, 900 µL MRS (Difco Laboratories) was adjusted to pH 2 or 4 or supplemented with 0.5% or 1% (w/v) Oxgall (Sigma-Aldrich). The medium without modifications was used as a control. An aliquot of 100 µL of a 24 h culture of L. fermentum ATCC 23271, previously washed with PBS, was inoculated into tubes containing modified or unmodified MRS. After incubation at 37 • C for 3 h under anaerobic conditions, the percentage of viable bacteria was calculated by counting CFUs on MRS agar (Difco Laboratories).

Ethical Aspects
Clinical microbial strains were obtained from a previous research project approved by the Ethics Committee of CEUMA University (N • 2.519.446/2018/CEP-UNICEUMA).

Statistical Analysis
Data analysis was performed using GraphPad Prism Software 5.1 (GraphPad Software, San Diego, CA, USA). Student's t-tests were performed to assess differences in the adhesion assays when the Candida strains were assayed alone or in the presence of L. fermentum ATCC 23271. All experiments were performed in triplicate on three independent days, and statistical significance was set at p < 0.05.

General Genome Features and Comparative Analysis
To search for the genetic basis of the probiotic properties of interest, the genome of L. fermentum ATCC 23271 was sequenced. The genome was deposited in the NCBI database under the BioProject accession number PRJNA729474. Raw reads were deposited in the Sequence Read Archive (SRA) under the accession number SRX10856814. The assembled genome sequence was deposited in DDBJ/ENA/GenBank under the accession number GenBank: JAHBRU000000000.1.
Preliminary annotation data for the L. fermentum ATCC 23271 genome are listed in Table 1. The genome contains 2123 protein-coding sequences (CDS). The RAST analysis showed the presence of 2123 coding sequences distributed in 312 subsystems, of which 1614 were related to non-hypothetical proteins and 509 to hypothetical proteins. These genes were found to be associated with several subsystems ( Figure 1). Through image analysis, it was seen that the subsystems with the highest number of genes are related to the production of cofactors, vitamins, prosthetic groups, pigments, protein metabolism, amino acids and carbohydrates. The genome of L. fermentum ATCC 23271 was compared with the whole genomes of other L. fermentum strains in GenBank using the OrthoVenn web platform. The species formed 2183 gene clusters, of which 1262 belonged to at least two species and 921 presented themselves in a single copy. It was also observed that L. fermentum ATCC 23271 The genome of L. fermentum ATCC 23271 was compared with the whole genomes of other L. fermentum strains in GenBank using the OrthoVenn web platform. The species formed 2183 gene clusters, of which 1262 belonged to at least two species and 921 presented themselves in a single copy. It was also observed that L. fermentum ATCC 23271 shares 941 orthological clusters with all the other tested strains, and that it has 13 exclusive gene clusters ( Figure 2).    Most proteins of these 941 clusters were found to be involved in the biological processes of chemical reactions and their pathways and cell division processes ( Figure 4).
With regard to molecular functions, 13.8% of the identified proteins are involved in hydrolase activity, 12.7% are related to a biochemical activity or are components of a larger process and 10% were responsible for the transport of substances inside, outside and between cells ( Figure 5).
The third group of proteins is related to the cellular components. In this group, most of the proteins comprised cell compartments ( Figure 6).         Using the reference genomes and other sequences available in the TYGS database, a phylogenetic tree was constructed ( Figure 7). Phylogenetic tree analysis showed that the ATCC 23271 strain was the first species to appear during the evolutionary process. Oenococcus alcoholitolerans CBAS 474 was used as the outgroup and, therefore, the most distant on the evolutionary scale among all analyzed species and strains. L. cellobiosus DSM 20055 was also found to be inserted within the clade with other L. fermentum strains. The clade formed by L. fermentum had a 97% confidence level. L. fermentum ATCC 23271 was found to be more closely related to L. cellobiosus DSM 20055, within the clade, whereas the nearest outside species of the clade was L. gorillae KZ01. Among the 13 exclusive clusters of L. fermentum ATCC 23271, OrthoVenn 2 analysis revealed 37 proteins that were most related to transposition. However, manual analysis showed only 16 ORFs, which included seven encoding transposases and six encoding hypothetical proteins, of which one sequence showed similarity to an L. reuteri hypothetical protein, two sequences that did not show similarity to any described protein and one en- Figure 7. Phylogenetic tree of Limosilactobacillus fermentum ATCC 23271 and other bacterial genera available in the TYGS database. The tree was inferred using FastME 2.1.6.1 from ribosomal DNA. Branch lengths are scaled using the GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values >60% of 100 replications, with an average branch support of 19.2%. The tree was rooted at the midpoint.
Among the 13 exclusive clusters of L. fermentum ATCC 23271, OrthoVenn 2 analysis revealed 37 proteins that were most related to transposition. However, manual analysis showed only 16 ORFs, which included seven encoding transposases and six encoding hypothetical proteins, of which one sequence showed similarity to an L. reuteri hypothetical protein, two sequences that did not show similarity to any described protein and one encoded a chloride transporter, as evidenced by UniProt analysis.

Putative Genes Associated with Probiotic Properties
Genes for the following probiotic features were searched within the genome: tolerance to stress conditions, production of lactic acids, production of adhesion structures, production of antimicrobial peptides and safety for human use. Genomic analysis detected 21 genes encoding proteins that may be related to the ability to tolerate the secretion of digestive enzymes, bile salts and acidic pH. Among them, genes for sugar metabolism and production of L-lactic acid were also present in the genome ( Table 2). Eight genes were related to proteins involved in bacterial ability to adhere to and colonize eukaryotic cells, thus competing with pathogenic microorganisms for cell receptors, including: three related to the aggregation process, four related to the production of exopolysaccharides and a fibronectin-binding domain-containing protein (Table 3). A search for genes related to the production of bacteriocins was performed using the BAGEL4 program. A sequence 48% similar to that of enterolysin A, a protein of Enterococcus faecalis, was obtained. Figure 8 shows the position of the bacteriocin sequence in green. A search for genes related to the production of bacteriocins was performed using the BAGEL4 program. A sequence 48% similar to that of enterolysin A, a protein of Enterococcus faecalis, was obtained. Figure 8 shows the position of the bacteriocin sequence in green. After identifying the cleavage site of the translated bacteriocin protein sequence, using the SignalP 5.0 server, the mature protein was analyzed in silico to predict its antimicrobial activity by using four algorithms, including Support Vector Machine (SVM), Random Forest (RF), Artificial Neural Network (ANN) and Discriminant Analysis (DA). Of these four algorithms, three indicated a high probability (>0.9) of the peptide to present antimicrobial activity (Table 4).  After identifying the cleavage site of the translated bacteriocin protein sequence, using the SignalP 5.0 server, the mature protein was analyzed in silico to predict its antimicrobial activity by using four algorithms, including Support Vector Machine (SVM), Random Forest (RF), Artificial Neural Network (ANN) and Discriminant Analysis (DA). Of these four algorithms, three indicated a high probability (>0.9) of the peptide to present antimicrobial activity (Table 4). In the genomic assessment of strain safety, the searches performed in both the Res-Finder and CARD databases did not identify any genes encoding acquired drug resistance. However, the L. fermentum ATCC 23271 genome possessed 19 genes related to intrinsic resistance to antibiotics and other toxic compounds, including heavy metals such as cobalt, mercury, cadmium, copper and zinc, or represent potential targets for antimicrobial resistance, such as penicillin-binding protein, elongation factor G, DNA topoisomerase IV subunit B, topoisomerase IV subunit A, DNA topoisomerase (ATP-hydrolyzing) subunit B and DNA gyrase subunit A (Table 5). In addition, the strain was predicted to be a non-human pathogen by the Pathogen-Finder tool hosted by the Centre for Genomic Epidemiology. The probability of being a human pathogen was calculated as 0.202, indicating a low probability for L. fermentum ATCC 23271 to present pathogenicity, and the estimated matched pathogenic families were 0. There was no hit for virulence determinants using the VirulenceFinder tool, also hosted by the Centre for Genomic Epidemiology. Two prophage regions were identified within the entire genome, and analysis using the PHASTER tool revealed that the sequences were incomplete. A search for the CRISPR-Cas sequence found two CDS putative sequences for the CRISPR sequences with the associated cas gene. These fragments occurred on contig identity NZ_JAHBRU010000076.1_1 (in the region between 17,626 and 18,783 bp) and NZ_JAHBRU010000115.1_1 (in the region between 850 and 3317 bp). The first identified CRISPR sequence contained 17 spacer genes and a 36 bp repeat consensus (GTCTTGGAT-GAGTGTCAGATCAGTAGTTCCGAGTAC), and the latter contained 40 spacer genes and a 28 bp repeat consensus (GGATCACCCCCATATACATGGGGAGCAC).

Antagonism Activity
To confirm the inhibitory activity of the ATCC 23271 strain against Candida spp. and its low activity against bacteria [37], the overlay method was used as an antagonism assay. L. fermentum ATCC 23271 impaired the growth of Candida spp. with inhibition zones ranging from 13.5 ± 2.1 mm to 26.5 ± 2.1 mm. The only exceptions were two Candida krusei strains (ATCC 6258 and GJFD), which were not inhibited by the probiotic. Regarding the assays with bacterial strains, inhibition was observed only in the area immediately above the growth of the ATCC 23271 strain, that is, without the formation of large inhibition zones (Table 6).

Interference in Pathogen Cell Adhesion
Due to the reduced inhibitory activity of L. fermentum on bacterial pathogens, interference assays on adhesion to HeLa cells were performed only with Candida strains. In the competition assay, three clinical strains (C. albicans CAS, C. krusei GJFD, and C. parapsilosis RCL) had their adherence to eukaryotic cells completely inhibited by L. fermentum ATCC 23271, an effect also noted for the reference strain C. parapsilosis ATCC 22019 ( Figure 9A). In the exclusion experiment, on one hand, L. fermentum ATCC 23271 decreased the adhesion of C. albicans ATCC 90028, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 and C. parapsilosis FSG to HeLa cells. On the other hand, C. krusei GJFD and C. albicans SC 5314 adhered more in the presence of L. fermentum ATCC 23271 ( Figure 9B). In the displacement assay, C. albicans CAS, C. krusei GJFD, C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 showed reduced adhesion to eukaryotic cells ( Figure 9C).

Tolerance to Bile Salts and Acidic pH
To assess L. fermentum ATCC 23271 resistance to adverse host conditions, the bacterium was incubated in the presence of bile salts and acidic pH. The microorganism demonstrated a higher tolerance when cultivated at pH 4.0; however, it still had a survival rate greater than 60% when in a more acidic pH (pH 2.0). Exposure to bile salts did not affect L. fermentum growth. On the contrary, the highest survival rate was found following

Tolerance to Bile Salts and Acidic pH
To assess L. fermentum ATCC 23271 resistance to adverse host conditions, the bacterium was incubated in the presence of bile salts and acidic pH. The microorganism demonstrated a higher tolerance when cultivated at pH 4.0; however, it still had a survival rate greater than 60% when in a more acidic pH (pH 2.0). Exposure to bile salts did not affect L. fermentum growth. On the contrary, the highest survival rate was found following incubation with 0.5% bile salts (Table 7).

Antibiotic Susceptibility Assay
Antibiotic susceptibility was assessed by the disc diffusion method. L. fermentum ATCC 23271 was susceptible to most of the tested antibiotics. L. fermentum ATCC 23271 also exhibited moderate susceptibility to cefoxitin and norfloxacin and resistance to gentamycin, trimethoprim/sulfamethoxazole and vancomycin (Table 8).

Discussion
This study presents for the first time, a genomic analysis of L. fermentum ATCC 23271 in regards to important characteristics which confer a strain, a probiotic profile. In addition to its in vitro anti-Candida activity, different in silico functional analyses have revealed several protein-coding sequences associated with other probiotic properties of L. fermentum ATCC 23271, as well as with its safety for human use.
The L. fermentum ATCC 23271 strain was subjected to comparative analysis using the OrthoVenn program with others of the same species, which have previously reported beneficial effects. In this context, L. fermentum CECT 5716 strain has been extensively studied, and its probiotic properties include modulation of the host immune response [58], enhanced response to influenza vaccination in adults [59], reduction of Staphylococcus load in breast milk of lactating mothers [60], and reduced incidence of respiratory and gastrointestinal infections in infants [32]. The DR 9 strain has demonstrated antioxidant effects (via upregulation of oxoproline) and immunomodulatory effects in aging rats [61,62]; it also prevents telomere shortening in aging rats [63]. The L. fermentum 3872 strain is known for its potential for combating Campylobacter jejuni infections [30]. This strain also has a unique collagen-binding protein encoded by the cbp gene with five repetitive "B domains" (whereas the other L. fermentum strains only have four "B domains") [64]. L. fermentum AGR1485 strain has the capacity to increase transepithelial electrical resistance (TEER) across Caco-2 cell layers, thus enhancing barrier integrity and protecting against infections by enteropathogens [65]. L. fermentum MCC 2760 has displayed several activities, including cholesterol reduction, high antioxidant potential, as well as anti-inflammatory actions [27,66]. Since L. fermentum ATCC 23271 shares 941 gene clusters with all those strains, it is possible that it also shares some of their probiotic properties.
Phylogenetic analysis showed that L. fermentum ATCC 23271 is part of the clade along with other strains of the same species, including L. cellobiosus DSM 20055. L. cellobiosus was first described by Rogosa et al. [67] and, like L. fermentum, is a heterofermentative Lactobacillus species. Both species share very similar phenotypic properties, and both belong to the subgenus 'Betabacterium' Orla-Jensen of Lactobacillus [68]. These authors support the idea that L. cellobiosus and L. fermentum should be united under the same name, with L. fermentum being the earlier synonym [68]. Previous studies have indicated a close relationship between L. cellobiosus and Limosilactobacillus fermentum; in fact, L. cellobiosus has now been reclassified as a biovar of L. fermentum [26]. The external lineage is L. gorillae KZ01, which is phylogenetically related to human-associated L. fermentum; however, it has not been found in humans yet and has been isolated from the feces of a captive western lowland gorilla (Gorilla gorilla) [69].
Concerning the tolerance to adverse conditions, the genome analysis showed that L. fermentum ATCC 23271 has several genes encoding proteins that are responsible for the resistance to acidic pH and bile salts. ATP synthases are some of the proteins involved mainly in acid tolerance, as they are associated with pH cytoplasmic regulation by ATP hydrolysis, which maintains pH homeostasis and protects cells from the damage induced by an acidic environment [70]. Glucose-6-phosphate isomerase is also involved in acid tolerance, acting as an acid shock protein [70]. All of these genes and others encoding proteins that promote acid and bile tolerance were detected in the genome of L. fermentum ATCC 23271 [71]. Phenotypic analysis also demonstrated its ability to survive in the presence of acidic pH and bile salts, corroborating its genetic background. In addition, L. fermentum ATCC 23271 contains putative genes encoding antioxidant enzymes, such as glutathione biosynthesis bifunctional protein, bifunctional glutamate-cysteine ligase, glutathione synthetase, peptide methionine sulfoxide reductases, free methionine-(R)-sulfoxide reductase, NADH peroxidase and thiol peroxidase. These enzymes (or their products) can protect cells against oxidative damage caused by reactive nitrogen intermediates and reactive oxygen species [55,72,73].
Additionally, other evidence shows that the enzyme methionine sulfoxide reductase (Msr) may also be involved in bacterial adhesion [74]. L. fermentum ATCC23271 has two types of this enzyme (MsrA and MsrB). In this regard, the ability to adhere to host tissues is an important attribute of probiotics [10]. L. fermentum ATCC 23271 presented nine genes encoding adhesion-related proteins, including exopolysaccharides (EPSs), which have different effects on probiotic adhesion to intestinal mucus, according to their different physicochemical and/or structural characteristics [75]. In addition, the gene that encodes the fibronectin-binding protein has also been found, and it is known to facilitate adhesion to the extracellular matrix of mammalian cells [76]. Both mechanisms could enable L. fermentum ATCC 23271 to adhere to host tissues and colonize the environment.
Competition for adhesion sites is an additional strategy used by probiotics to inhibit colonization by pathogens. In the present study, a strong ability to inhibit the adhesion of some Candida strains to eukaryotic cells was observed (mainly genital clinical isolates) in the competition and displacement assays. Heinemann et al. [77] showed that L. fermentum RC-14 is capable of releasing an active surface component, which can inhibit the adhesion of uropathogenic bacteria. In the same study, it was possible to purify a protein with anti-adhesive capacity against Enterococcus faecalis 1131. Another study showed that L. fermentum isolated from humans inhibits the adhesion of enteropathogens, such as that of E. coli to host cells, through an SAP protein [78]. The fact that some pathogens present greater adhesion in the presence of L. fermentum ATCC 23271 may be related to the production of aggregation proteins or EPS, as previously suggested [75,79,80].
Bacteriocins are small peptides secreted by many Gram-positive bacteria, with significant activity against distinct microorganisms [81]. A recent study demonstrated that SD11, an L. fermentum-derived bacteriocin, possesses anti-Candida activity [82]. Herein, genomic analysis of L. fermentum ATCC 23271 by the BAGEL 4 program showed the presence of a gene encoding a hypothetical protein with low similarity (48%) to enterolysin A. Interestingly, this bacteriocin is known to be produced by Enterococcus faecalis LMG 2333 and to inhibit some species of Enterococcus, Lactobacillus, Lactococcus and Pediococcus [83]. We also demonstrated that three algorithms indicated a high probability of this protein with antimicrobial activity. The evidence allows us to suggest that the antifungal activity observed in our study may be due to bacteriocins. However, further studies are necessary to determine the exact bacteriocins or any additional proteins involved in this activity.
Resistance to antibiotics is a concern because of the possibility of transferring the plasmid containing these genes to other pathogenic bacteria, making infections difficult to treat. The European Food Safety Authority (EFSA) recommends that bacterial strains harboring transferable antibiotic resistance genes should not be used as probiotics in animal feeds, fermented foods, and foods for human consumption [84]. Lactobacilli are susceptible to all protein synthesis inhibitors except aminoglycosides but are generally intrinsically resistant to quinolones, trimethoprim and sulphonamides, as well as, in the case of L. fermentum, also to glycopeptides [85]. All antibiotics to which L. fermentum ATCC 23271 showed resistance or moderate susceptibility indicate an intrinsic rather transferable resistance capacity.
L. fermentum ATCC 23271 presented two regions of prophages found in the genome characterized as incomplete, indicating that they were not functional. The identified transposases and other insertion sequences did not flank the resistance genes, further limiting their transferability. Furthermore, genome analysis revealed two regularly intercalated short palindromic repeat sequences (CRISPR) with the Cas gene and associated spacers. It has been reported that the presence of a CRISPR region may limit the spread of antimicrobial-resistant genes by inhibiting gene transfer pathways [86]. The CRISPR-Cas fragments work as a line of defense for the host strain against the insertion of extrachromosomal DNA molecules [50,86]. Therefore, their presence in the strain ATCC 23271 suggests a reduced probability of acquiring antimicrobial-resistant genes.
The protective role of Lactobacillus spp. against Candida spp. has been controversial, as the microorganism can be observed in high amounts in patients with Candida vaginitis [87]. In addition, many women with candidiasis may not have an altered microbiota [88]. These differences may be due to several factors including those inherent to the patient (such as age, immune status and symptoms) and factors related to the virulence properties of the Candida species causing the infection [89]. On the other hand, it has already been clearly demonstrated that a diversity of Lactobacillus spp. has antifungal effects [90][91][92]. Furthermore, only some strains of Lactobacillus can produce antimicrobial compounds in the amounts necessary for an antifungal activity, which possibly explains the failure of some vaginal lactobacilli to suppress colonization by Candida. In this context, our data indicate that L. fermentum ATCC 23271 may represent a potential strategy to prevent Candida colonization.
Genome sequence analysis can contribute to the understanding of the molecular basis of the probiotic functions of L. fermentum ATCC 23271. However, the genetic basis for growth and adhesion inhibitory activities against Candida need to be validated. Thus, gene knockout mutants of this strain are now being constructed to better assess the roles of the putative antimicrobial peptide and adhesin genes in these activities. The most notable feature is the number of genes associated with strain adhesion and survival under unfavorable conditions such as acidic pH, bile salts or even oxidative environments. Other interesting genomic components that represent some of the features that contribute to probiotic activity include many genes involved in sugar transport and metabolism, including oligosaccharides. One element that is part of the general characteristics of a probiotic is its safety for human use, which is assured by the absence of acquired antibiotic resistance and virulence genes, and the presence of gene loci associated with CRISPR/CRISPR (cas) and incomplete prophage regions.

Conclusions
The data from the present study demonstrate that L. fermentum ATCC 23271 is a probiotic candidate with anti-Candida activity. It can inhibit the growth of C. albicans and C. parapsilosis, in addition to being able to interfere with the adhesion of Candida spp. to host cells. Genomic analyses showed a variety of genes possibly associated with strain adhesion to host cells and molecules, tolerance to acidic pH, bile salts and oxidative stress as well as safety for human consumption. A limitation of the study is that the mechanism of growth inhibition of different Candida species is yet to be completely established. Although we have identified a probable protein with antifungal activity, we still need to validate this finding through studies with mutants, with synthetic peptides (whose sequences are derived from this protein) and also in animal experiments. An important aspect revealed by the comparative genomic analysis is that the strain shares many genes with other strains of the same species that have different probiotic properties. Nonetheless, our findings indicate a promising use of L. fermentum ATCC 23271 as an anti-Candida therapy.