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Article

Characterization and Probiotic Potential of Levilactobacillus brevis DPL5: A Novel Strain Isolated from Human Breast Milk with Antimicrobial Properties Against Biofilm-Forming Staphylococcus aureus

by
Ivan Iliev
1,*,
Galina Yahubyan
2,
Elena Apostolova-Kuzova
2,
Mariyana Gozmanova
2,
Daniela Mollova
1,
Iliya Iliev
1,
Lena Ilieva
1,
Mariana Marhova
1,
Velizar Gochev
1 and
Vesselin Baev
2,*
1
Department of Biochemistry and Microbiology, Faculty of Biology, University of Plovdiv, Tzar Assen 24, 4000 Plovdiv, Bulgaria
2
Department of Molecular Biology, Faculty of Biology, University of Plovdiv, Tzar Assen 24, 4000 Plovdiv, Bulgaria
*
Authors to whom correspondence should be addressed.
Microorganisms 2025, 13(1), 160; https://doi.org/10.3390/microorganisms13010160
Submission received: 23 December 2024 / Revised: 8 January 2025 / Accepted: 11 January 2025 / Published: 14 January 2025
(This article belongs to the Special Issue Beneficial Microorganisms and Antimicrobials: 2nd Edition)
Browse Figures
Figure 1
<p>Optical density (600 nm) after culturing in the presence of different sugars in a <span class="html-italic">Lactobacillus brevis</span> DPL5.</p> "> Figure 2
<p>Effect of different concentrations of sodium chloride on the growth of <span class="html-italic">Levilactobacillus brevis</span> DPL5.</p> "> Figure 3
<p>Survival of <span class="html-italic">L. brevis</span> DPL5 in different acidic conditions.</p> "> Figure 4
<p>Antagonistic activity against nasal <span class="html-italic">Staphylococcus aureus</span> strain: Plug diffusion test with 24 h (<b>A</b>), 48 h (<b>B</b>), and 72 h (<b>C</b>) <span class="html-italic">L. brevis</span> DPL5 cultivated on MRS agar. Agar well diffusion test showing the activity of <span class="html-italic">L. brevis</span> DPL5 cell-free supernatant (50 µL) after anaerobic cultivation in MRS broth for 24 h (<b>D</b>) and 48 h (<b>E</b>).</p> "> Figure 5
<p>Effect of different concentrations of lyophilized <span class="html-italic">L. brevis</span> DPL5 cell-free supernatant on the intensity of biofilm formation in nasal <span class="html-italic">S. aureus</span> strains.</p> "> Figure 6
<p>Confirmation of the <span class="html-italic">L. brevis</span> DPL5 via genome-to-genome comparisons in TYGS.</p> "> Figure 7
<p>Genomic maps of the clusters of the lanthipeptide class IV, T3PKS region, and RiPP-like region of the <span class="html-italic">L. brevis</span> DPL5.</p> ">
Review Reports Versions Notes

Abstract

:
Lactobacillus is a key genus of probiotics commonly utilized for the treatment of oral infections The primary aim of our research was to investigate the probiotic potential of the newly isolated Levilactobacillus brevis DPL5 strain from human breast milk, focusing on its ability to combat biofilm-forming pathogens such as Staphylococcus aureus. Employing in vitro approaches, we demonstrate L. brevis DPL5′s ability to endure at pH 3 with survival rates above 30%, and withstand the osmotic stress often found during industrial processes like fermentation and freeze drying, retaining over 90% viability. The lyophilized cell-free supernatant of L. brevis DPL5 had a significant antagonistic effect against biofilm-producing nasal strains of Staphylococcus aureus, and it completely eradicated biofilms at subinhibitory concentrations of 20 mg·mL−1. Higher concentrations of 69 mg·mL−1 were found to have a 99% bactericidal effect, based on the conducted probability analysis, indicating the production of bactericidal bioactive extracellular compounds capable of disrupting the biofilm formation of pathogens like S. aureus. Furthermore, genome-wide sequencing and analysis of L. brevis DPL5 with cutting-edge Nanopore technology has uncovered over 50 genes linked to probiotic activity, supporting its ability to adapt and thrive in the harsh gut environment. The genome also contains multiple biosynthetic gene clusters such as lanthipeptide class IV, Type III polyketide synthase (T3PKS), and ribosomally synthesized, and post-translationally modified peptides (RiPP-like compounds), all of which are associated with antibacterial properties. Our study paves the way for the further exploration of DPL5, setting the stage for innovative, nature-inspired solutions to combat stubborn bacterial infections.

1. Introduction

Human breast milk is a multifaceted and adaptive substance that represents the pinnacle of infant feeding. It offers the ideal nutrient content, immunological elements, and bioactive constituents essential for the growth, development, and safeguarding of the newborn [1,2]. There is increasing emphasis on the importance of human breast milk as a source of beneficial bacteria that contribute to a healthy gut microbiome in babies [3]. We acknowledge it as an excellent source of probiotics because the bacteria it contains fulfill essential criteria for probiotics: human origin, adaptability to milk substrates, and a documented history of safe, extended consumption by newborns [4,5]. Numerous bacterial families, such as Streptococcaceae, Pseudomonadaceae, Staphylococcaceae, Lactobacillaceae, and Oxalobacteraceae, are prevalent in human breast milk [6,7,8]. Lactobacillus and Bifidobacterium, notable genera among the several bacteria species present in human breast milk, are recognized for their probiotic potential [9,10,11]. Research indicates that breast milk serves as a natural reservoir for multiple species of Lactobacillus, including L. rhamnosus, L. plantarum, L. fermentum, L. gasseri, L. salivarius, L. reuteri, L. casei, and L. brevis [9,10,12,13].
Lactic acid bacteria (LAB) are Gram-positive, catalase-negative, non-spore forming, coccus- or rod-shaped bacteria that produce organic acids after glucose fermentation [11,14,15]. Although most probiotics belong to the LAB family, not all group members possess probiotic properties. As established by the World Health Organization (WHO), probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [16]. For an LAB to be acknowledged as a probiotic, it must survive the harsh conditions of the gastrointestinal tract. Probiotic bacteria can produce antimicrobial substances, adhere to intestinal cells, and inhibit the adhesion of pathogenic bacteria, thereby ensuring their survival [17,18] Numerous Lactobacillus strains produced from human breast milk have been employed as probiotics, exhibiting effectiveness in preventing and treating infections, inflammation, and allergies [19]. Due to their probiotic properties, several strains have been generally recognized as safe by the Food and Drug Administration and qualified presumption of safety by the European Food Safety Authority [20].
Many studies have demonstrated that Lactobacillus species may produce lactic acid, hydrogen peroxide, and bacteriocins, among other antimicrobial compounds, which can hinder harmful bacterial strains’ growth and biofilm formation. Bacteriocins are antimicrobial peptides produced by bacteria, including Lactobacillus, and display bacteriostatic or bactericidal activity against other bacteria [21]. Ribosomes manufacture bacteriocins. These peptides disrupt the cell membrane of target bacteria, leading to cell death. Studies have demonstrated that Lactobacillus-derived bacteriocins and other mechanisms of action, such as competition for adhesion sites and nutrients, act as effective inhibitors of growth and biofilm formation in Staphylococcus aureus [22,23].
S. aureus is a Gram-positive bacterium that is widespread in the environment and frequently colonizes humans’ skin and mucous membranes. It is a major human pathogen capable of causing many infections, ranging from mild skin infections to life-threatening conditions such as pneumonia, endocarditis, and sepsis [24,25]. The ability of S. aureus to form biofilms, complex microbial communities attached to surfaces, contributes to its virulence and antibiotic resistance. Biofilms provide a protective barrier for bacteria, making them more difficult to eradicate with conventional antibiotics [26,27].
Despite the limited research on the probiotic potential of L. brevis from human breast milk, existing studies suggest that using Lactobacillus and its bacteriocins as natural antimicrobial agents to control S. aureus infections is promising, and it could help cut down on antibiotic use and the development of antibiotic resistance [28]. The study aims to look at the genomic structure and probiotic potential of L. brevis from human breast milk and evaluate its antibacterial properties against S. aureus. Specifically, this research seeks to evaluate the antimicrobial activity of L. brevis-derived cell-free supernatant and its ability to prevent biofilm formation. By exploring the interaction between Lactobacillus metabolites and S. aureus, this study aims to contribute to the growing body of knowledge surrounding the use of probiotics and their metabolites in managing bacterial infections, particularly those involving biofilm formation.

2. Materials and Methods

2.1. Breast Milk Sample Collection

Breast milk samples were collected from nursing mothers within the first six months postpartum. All participants gave informed consent for their inclusion. The research was conducted by the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee at the University of Plovdiv (Approval No. 6/06.10.2022). The participants were healthy women who had delivered full-term infants either via vaginal birth or cesarean section. The mothers were asked to wash their breasts with water to collect the milk samples and then express 15–20 mL of milk into a sterile container. These containers were stored at 4 °C until retrieved and transported to the laboratory. All milk samples were processed within 24 h of collection.

2.2. Bacterial Isolation and Identification and Culture Conditions

Standard laboratory protocols were followed to collect and identify LAB [2]. At first, samples with 1 mL volume were enriched by 24 h anaerobic incubation in 9 mL De Man Rogosa and Sharpe (MRS) liquid medium. Tenfold serial dilutions were spread plated on De Man Rogosa and Sharpe (MRS) agar and incubated anaerobically at 37 °C for 72 h in a 2.5 L anaerobic jar with Anaerocult A reagent (Merck, Darmstadt, Germany). Single colonies that developed on the MRS agar were purified by streaking on new dishes with MRS agar [2]. The preliminary identification of Lactobacillus strains included Gram staining and test for catalase production. According to Bergey’s Manual of Systematic Bacteriology, bacterial isolates were initially identified based on their colony morphology, cultural characteristics, and microscopic appearance [29]. Those determined to be Gram-positive, non-spore-forming, and catalase-negative rods were presumptively identified as Lactobacillus spp. The isolated strains were preserved as stock cultures at −20 °C in MRS broth (Merck, Darmstadt, Germany) with 15% glycerol (Merck, Darmstadt, Germany) for further analysis. Species identity was confirmed by GeN III plate with Biolog’s OmniLog ID System (Swampscott, MA, USA) instrument [30]. Isolate DPL5, identified as Lactobacillus brevis, was chosen for further analysis.
Staphylococcus aureus strains used for the inhibitory tests were obtained from the Microorganisms Culture Collection of the Department of Biochemistry and Microbiology (Paisii Hilendarski Plovdiv University, Plovdiv, Bulgaria). A total of 33% were MRSA strains to detect differences in the effects of L. brevis on cell proliferation and biofilm formation. Information regarding the antimicrobial susceptibility of the isolates is presented in Table S1. The strains were cultured in tryptic soy broth (TSB) supplemented with 1% (w/v) glucose and 2% (w/v) NaCl [31]. S. aureus inoculums were standardized by homogenization in sterile 0.85% NaCl, and all suspensions were diluted to 0.5 × 108 CFU mL−1 using McFarland densitometer DEN-1 (Grant-bio, Cambridge, England) prior to inoculation [24].

2.3. Probiotic Potential Assessment

2.3.1. Assimilation of Different Types of Carbon Sources and Osmotic Sensitivity

The metabolic profile of L. brevis DPL5 was analyzed using Biolog’s Phenotype MicroArrays™ (PM) in conjunction with the Omnilog™ (Orlando, FL, USA) instrument. During the experiment, the samples were incubated and read continuously at 20-min intervals for 24 h in the Omnilog instrument. Data collection involved a two-step approach. First, Omnilog was used to measure the OD in each well and directly assess cell proliferation. A redox-sensitive dye was used to measure the color change in each well based on NADH production, allowing the determination of metabolic activity. The simultaneous measurements of both cell density and color change in the dye make it possible to understand the differences between metabolism and cell growth in terms of their phenotypic differences. A Gen III plate with inoculation fluid A was used in the experiments.

2.3.2. Bile Salt Tolerance

The strain’s capacity to endure various amounts of bile salts was estimated at three distinct concentrations of bile salts: 0.3%, 1%, and 3%. MRS with bile salts was prepared and inoculated with overnight bacterial suspension (dilution factor 10). Optical density was assessed 24 h post-inoculation at a wavelength of 600 nm using the Beckman Coulter DU 730 spectrophotometer (Brea, CA, USA), and bacterial growth levels were documented [31]. The assay was performed twice each in triplicate.

2.3.3. Acid Tolerance

The capacity to endure acidic conditions was evaluated by exposing samples to pH 3, 5, 7, 9 and 10 in MRS broth inoculated with overnight bacterial suspension (dilution factor 10). Optical density was assessed 24 h post-inoculation at a wavelength of 600 nm with the Beckman Coulter DU 730 spectrophotometer (Brea, CA, USA). The readings were recorded at 24 h of incubation, and this process was repeated twice for each sample.

2.3.4. Determination of Antibiotic Susceptibility

The method described by Sharma and collaborators [32] was used to evaluate the antibiotic susceptibility of the L. brevis DPL5. The susceptibility to ten commonly used clinical antibiotics (Erythromycin, Amikacin, Gentamicin, Kanamycin, Amoxicillin, Ampicillin, Penicillin, Ciprofloxacin, Chloramphenicol, and Ceftriaxone) was determined using the disc diffusion method. Antibiotics were obtained from Oxoid (Thermo Fisher Scientific, Wesel, Germany). Active cultures were prepared by adjusting the suspension densities to McFarland 0.5. Then, 100 µL of the isolate was spread onto MRS agar (Merck, Darmstadt, Germany). Subsequently, three antibiotic discs were placed on inoculated MRS agar and incubated at 37 °C for 24 h. After incubation, the diameter of the zone of inhibition around each disc was measured. The zone of inhibition represents the area where the growth of the microorganism was restrained, following the guidelines of the Clinical and Laboratory Standards Institute [33]. The multiple antibiotic resistance (MAR) index was calculated using the method of Gyorgy et al. [34].

2.4. Preparation and Lyophilization of L. brevis Cell-Free Supernatant (LCFS)

Lactobacillus brevis DPL5 was cultured in 200 mL MRS broth for 48 h. The cells were separated by centrifugation at 10,000× g for 20 min at 4 °C. The supernatant was filtered through a 0.22 µm nitrocellulose membrane. To eliminate the effect of lactic acid, the pH of the supernatant was neutralized [35]. The sterile supernatant and pure MRS medium were frozen at −80 °C and lyophilized (Labcocno FreeZone 4.5; Labconco Corporation, Kansas City, MI, USA)). The lyophilized samples were weighted and stored at −20 °C [31]. Deionized water was used to rehydrate to the desired concentration prior to use.

2.5. Antagonistic Activity Against S. aureus

The antagonistic activity tests of L. brevis DPL5 against 30 pathogenic S. aureus strains were evaluated using an agar plug diffusion test [36,37]. Briefly, 24 h liquid culture of L. brevis DPL5 was streaked on two MRS agar plates and incubated anaerobically at 37 °C for 24 and 48 h in 2.5 L anaerobic jar with Anaerocult A reagent (Merck, Darmstadt, Germany). Overnight cultures of the tested S. aureus strains were diluted to 1.5 × 108 CFU mL−1 and uniformly spread using a sterile cotton swab on Mueller Hinton Agar plates (Merck, Darmstadt, Germany). The plates were dried for 30 min at room temperature. Then, 0.25 cm2 agar plugs of 24 h and 48 h L. brevis cultures were cut out and placed on the previously streaked S. aureus plates. Following 24 h incubation at 37 °C, the antagonism was determined by the presence/absence of inhibition zones around the agar plugs. The agar well diffusion method was used to determine the activity of L. brevis DPL5 cell-free supernatant [38]. L. brevis DPL5 was cultivated in 100 mL MRS broth anaerobically for 24 h and 48 h, respectively. The cultures were centrifuged, and the supernatants were filtered through a syringe filter with a pore size of 0.22 µm. As described previously, a 50 µL cell-free supernatant was added into separate, 6 mm diameter holes in the agar gel of plates inoculated with S. aureus.

2.6. Minimum Inhibitory Concentration (MIC) Assay

MIC assays with LCFS were performed by microdilution in 96-well plates (Costar®, Corning, NY, USA) [39]. A serial dilution was performed starting with 80 μg∙mL−1 of the supernatant on Tryptic Soy Broth, supplemented with 1% (w/v) glucose, 2% (w/v) NaCl, and 3% human plasma (TSBGSP), containing 5 × 105 CFU∙mL−1 of S. aureus per well [31]. The results were compared against a lyophilized medium (MRS) without L. brevis DPL5. TSBGSP without inoculum was used as negative (sterility) control, and TSBGSP containing 5 × 105 CFU∙mL−1 of S. aureus was used as positive control. Each S. aureus strain was tested in triplicates. The microplates were incubated for 24 h at 37 °C. The optical density of each well was determined in a spectrophotometer Multiskan FC (Thermo Scientific, Shanghai, China) at 620 nm. The absorbance was corrected against the negative control. MIC was determined as the lowest concentration, which inhibits the growth of S. aureus. Then, 5 μL from the MIC well and the wells with the next two higher concentrations were spotted on a new sterile MH agar plate and incubated for 24 h at 37 °C to classify the inhibitory concentrations as bactericidal or bacteriostatic.

2.7. Microtiter Plate Assay and Anti-Biofilm Activity

The quantitative test for biofilm formation was conducted using tissue culture polystyrene 96-well microtiter plates with flat bottoms (Costar®, Corning, NY, USA), following the methodology outlined by Čuvalová and Kmet [25] with minor changes. Staphylococci were cultured on MH agar (Merck), and single colonies were subsequently transferred to TSBGSP to achieve a concentration of 1.5 × 108 CFU.ml−1. The effect of LCFS on staphylococcal biofilm formation was tested by the addition of 100 μL of different concentrations of supernatant to the polystyrene microtiter plates. Eight serial dilutions were prepared, yielding concentrations of 160, 80, 40, 20, 10, 5, 2.5 and 1.25 mg∙mL−1. The cell density of S. aureus strains was adjusted to 1 × 106 CFU.ml−1, and 100 μL was dispensed into the preloaded with 100 μL LCFS microplate wells to a final concentration of 0.5 × 105 CFU.ml− and a final volume of 200 μL. The plates were incubated statically for 24 h at 37 °C. After incubation, the plates were washed thrice with 250 μL of saline solution (0.85% NaCl). Adherent cells were subjected to staining with a 0.1% crystal violet solution (Himedia, Dindhori, India) for 10 min. The surplus stain was removed by filling the wells with a saline solution (0.85% NaCl). The adherent dye was solubilized with 70% ethanol. The optical density of the wells was assessed at 610 nm utilizing a Multiskan FC (Thermo Scientific, Shanghai, China). Control wells contained 100 μL culture medium and 100 μL of the tested strain without adding CFLS. After 24 h of incubation at 37 °C, biofilm was quantified in the same manner as described above. As described by Saidi and collaborators [30], the Optical Density (OD) of the negative control well was recorded as ODc, and the OD of tested wells as ODt. The positive control tests with S. aureus demonstrated that all 30 strains had an ODt/ODc ratio > 4, classifying them as strong biofilm producers.

2.8. DNA Extraction, Sequencing, and Assembly

DNA was extracted from isolate DPL5 using the QIAamp DNA Microbiome Kit (QIAGEN, Hilden, Germany). The concentration and quality of the extracted DNA were evaluated using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis. To prepare long-read Oxford Nanopore Technology (ONT) libraries, the Ligation Sequencing Kit SQK-RBK114 (Oxford Nanopore Technologies, Oxford, UK) was utilized with 200 ng of total DNA following the manufacturer’s protocol. Subsequently, the prepared library underwent sequencing on a MinION device using an R10 flow cell (Oxford Nanopore Technologies, Oxford, UK). Base calling and quality checks were performed offline via Guppy v6.5.7 (Oxford Nanopore Technologies, Oxford, UK). Adapter sequences were removed using Porechop v0.2.4 with default settings (https://github.com/rrwick/Porechop, accessed on 10 January 2025).
De novo assembly was executed with Flye v2.9.2 under default settings, excluding reads shorter than 1000 bp. Polishing of the assembly was completed using Racon v1.4.21 (https://github.com/isovic/racon, accessed on 10 January 2025) and Medaka v1.8.1 (https://github.com/nanoporetech/medaka, accessed on 10 January 2025). The quality of the final assembly was verified using CheckM v1.1.6. A circular genome map of the single circular chromosome contig was visualized using the Proksee software (https://proksee.ca/, accessed on 10 January 2025).

2.8.1. Genome-Based Identification and MultiLocus Sequence Typing (MLST)

The bacterial species identification of isolate DPL5 was achieved by calculating its average nucleotide identity (ANI) using FastANI. Additionally, the genome was analyzed for MLST using PubMLST (https://pubmlst.org, accessed on 10 January 2025). The Type (Strain) Genome Server (TYGS) was also utilized to construct a whole-genome sequence-based phylogenetic tree (https://tygs.dsmz.de/, accessed on 10 January 2025).

2.8.2. Genome Annotation

The assembled genome of the DPL5 strain was submitted to NCBI Genomes for initial annotation through the Prokaryotic Genome Annotation Pipeline (PGAP) [40], which also assigned an accession number. The GenBank file generated by PGAP was further annotated using the Rapid Annotations using Subsystems Technology (RAST) [41] web server. Functional annotations were enhanced using the KEGG database and the BlastKOALA [42] tool by analyzing the predicted protein sequences from the PGAP GenBank file. The manual curation of genes related to probiotic properties was conducted using both RAST and KEGG-derived annotations. Carbohydrate-active enzymes (CAZymes) in the PU3 genome were identified using the cbCAN3 tool and the CAZy database (https://bcb.unl.edu/dbCAN2/, accessed on 10 January 2025). The genome of DPL5 was screened for antimicrobial resistance (AMR) and virulence factor (VF) genes using the Abricate tool (https://github.com/tseemann/abricate, accessed on 10 January 2025) with default settings, referencing the Comprehensive Antibiotic Resistance Database (CARD) [43], MEGARes DB [44], and the Virulence Factor of Bacterial Pathogens database (VFDB) [45]. Bacteriocin-related regions were predicted using the AntiSMASH 7.0 tool (https://antismash.secondarymetabolites.org/, accessed on 10 January 2025).

2.9. Statistical Analysis

Analysis of variance, t-test, graphical representation, and other results analyses were performed using Statistica 12 (StatSoft). The data for L. brevis are represented as mean ± standard deviation (SD). The data for biofilm formation in this study are expressed as mean ± 95% confidence intervals (p < 0.05) from the results of the 30 tested S. aureus strains. Probit analysis was performed using SPSS version 23 to estimate the MIC and minimal biofilm inhibitory concentration probability. All measurements were carried out in triplicate.

3. Results

3.1. Assimilation of Different Types of Carbon Sources and Osmotic Sensitivity

Lactobacilli have intricate nutritional needs for fermentable carbohydrates and derive energy through homofermentative or heterofermentative carbohydrate fermentation [46]. Their wide ecological distribution and adaptability to diverse habitats highlight their metabolic versatility, enabling the utilization of a broad range of carbohydrates [47]. Carbon catabolite repression (CCR) plays a key role in the competitive fitness of lactobacilli in natural environments like the gastrointestinal tract (GIT), where selecting the preferred carbon source significantly influences the growth rate and competition with other microorganisms [48]. We followed the uptake of different sugars in the Lactobacillus brevis DPL5 strain we studied. The results are presented in Figure 1. The analysis used the Biolog system and specific plates containing the studied sugars. We found good optical density in the medium’s presence of sugars such as lactose, sucrose, trehalose, and fucose. The strain has difficulty digesting sugars such as inosine, rhamnose, N-acetyl-D-mannosamine, N-acetyl-neuraminic acid, N-acetyl-D-galactosamine, and others.
L. brevis is frequently found in plant matter, but it has also been isolated from various other environments, such as beverages and the intestinal tracts of animals. This species is obligately heterofermentative, employs the phosphoketolase pathway, and possesses inducible glycolytic enzymes [49]. L. brevis has been shown to transport glucose, lactose, xylose, and galactose via proton symport systems. These proton symport systems are regulated through HPr, in which the transport mechanism is reversibly switched between proton symport and facilitated diffusion [50].
During industrial processes such as fermentation and freeze drying, lactic acid bacteria (LAB) often face significant osmotic stress [51]. Lactic acid bacteria primarily adapt to increased osmotic pressure through a two-phase response involving the accumulation of potassium ions (K+) and their counterions, such as proline and glycine betaine [52]. According to Sleator and Hill [53], the primary role of K+ accumulation in Gram-positive bacteria is to act as a signal, triggering the buildup of intracellular amino acids. We investigated the effect of different concentrations of sodium chloride −1%, 4%, and 9% on the growth of the Lactobacillus strain (Figure 2). No significant differences were established for the samples cultivated in 1% and 4% NaCl (p > 0.05). However, the highest concentration inhibited cell density for L. brevis DPL5 (p < 0.05).

3.2. Bile Salt Tolerance

The Food and Agriculture Organization and the World Health Organization define probiotics as “live microorganisms that, when consumed in sufficient quantities, provide health benefits to the host.” For probiotics to influence the intestinal environment, their population must reach a minimum threshold of 106 to 108 CFU per gram of intestinal content [54]. To establish themselves in the intestinal tract, these microorganisms must first withstand the harsh conditions of the digestive system, such as the acidic pH of the stomach and the presence of bile in the intestines. This ability to survive and thrive in such environments is a key characteristic of probiotics. In this regard, we investigated the influence of different percentage concentrations of bile salts added to the medium and followed the growth of the test strain L. brevis DPL5—Table 1.

3.3. Acid Tolerance

LAB employ various strategies to tolerate acidic conditions. These include producing alkaline substances via the arginine dihydrolase system to counteract acidity, neutralizing protons with carbon dioxide generated through malolactic fermentation, and expelling protons using proton pumps such as F1-F0-ATPase [55].
Figure 3 illustrates the cell viability of L. brevis DPL5 under various conditions. The viability of the cells at lower pH values is higher in the presence of low concentrations of pepsin in the medium compared to the viability that we reported in the absence of the enzyme. At pH around 5 and 7, no significant difference in survival is found in either variant.
Interestingly, pepsin demonstrated varying effects on cell viability. While pepsin is generally recognized for reducing microorganism viability through its proteolytic activity [56], exposure to pepsin increased the viability of strain DPL5 cells (Figure 3). This finding aligns with a previous study showing an increased viability of Bifidobacterium animalis subspecies upon exposure to pepsin. Although the precise mechanisms by which pepsin enhances the acid tolerance of lactic acid bacteria remain unclear, earlier research suggested that pepsin might aid in maintaining pH balance by supporting the activity of H+-ATPase in Bifidobacterium animalis subsp. lactis. This is thought to involve pepsin enhancing the proton pump’s function through ATP production [57]. While this hypothesis is yet to be validated, our findings support a similar explanation. Surviving the highly acidic environment of the gastrointestinal tract (GIT) is the first hurdle probiotic strains face, as gastric acid in the human stomach maintains a pH of approximately 1.5–3 [58]. To endure these extreme conditions in the gut, probiotic strains must possess acid tolerance. Many probiotics, particularly lactic acid bacteria (LAB), exhibit this tolerance and are essential in the fermentation industry [59]. Fermented food products are typically produced under specific acidic conditions facilitated by probiotic bacteria or yeast [60]. LAB such as Lactobacillus brevis, Lactobacillus fermentum, and Leuconostoc mesenteroides are used in fermenting durian fruit [61].

3.4. Determination of Antibiotic Susceptibility

The antibiotic susceptibility of L. brevis DPL 5 was assessed using the test paper agar diffusion method, and the findings are presented in Table 2. The strain demonstrated sensitivity to antibiotics such as tetracyclines, cephalosporins, and β-lactamase inhibitor complexes while showing resistance to Amikacin and Ceftriaxon. Aminoglycoside antibiotics inhibit protein synthesis and are known for their stability and broad-spectrum activity, primarily targeting Gram-negative bacteria while being less effective against Gram-positive bacteria. Experimental results for L. brevis DPL5 aligned with the previous literature [62].
The established resistance to ceftriaxone raises some concerns about the possibility for L. brevis DPL5 to act as reservoirs of transferable antibiotic resistance genes, but such resistance is not untypical [63]. Reports in recent years demonstrate that strains belonging to the Lactobacillus brevis group could often display high MIC values against β-lactams [64], and a wide occurrence of resistance to fourth and third-generation cephalosporins has been found even for other commercially available probiotic strains [65,66]. Moreover, the antibiotic resistance of lactobacilli can be considered ambivalently, as such resistance favors their survival during antibiotic therapy [67].

3.5. Antagonistic Effect of L. brevis DPL5 Using Plug-Diffusion and Well-Diffusion Methods

A preliminary antibacterial test was conducted to minimize time expenses. Due to the significant differences in the optimal culture conditions, the standard spot-on-the-lawn assay was ineffective for testing the possible inhibition effects of L. brevis DPL5 on Staphylococcus aureus. Instead, plug diffusion was used to test the antagonism [68]. Figure 4A indicates that the anaerobically cultured DPL5 for 24 h successfully combated S. aureus. The effects were probably mediated by direct cell competitive exclusion, as stated by Sikorska and Smoragiewicz [69]. The diameter of the inhibition zone (IZ) depends on the cultivation time. Increasing the cultivation time shows a positive effect with the highest IZ observed when 48 h culture DPL5 was applied (Figure 4B). Our findings paralleled the literature where [70] lactobacilli reach the maximum level of antimicrobial compound production during the stationary phase of their growth curve. Additional cultivation for up 72 h leads to a decrease in the antimicrobial activity (Figure 4C).
An additional well-diffusion assay with cell-free supernatant was performed to test the bioactivity of the extracellular compounds. Contrary to other studies [71], it shows that DPL5 possesses a better supernatant activity than cell-on-cell activity. The CFS significantly inhibited the growth of S. aureus. Results showed that the inhibition zones were larger when LCFS at 48 h of cultivation was used (21 mm) rather than at 24 h (11 mm) (Figure 4D,E). No significant differences in the diameter of IZ between MRSA and MSSA strains (p = 0.814) were found. The findings agree with several previous studies demonstrating the antimicrobial activity of lactobacilli [72] and their LCFS [73,74,75].

3.6. Antimicrobial Activity of DPL5

Antimicrobial activity is crucial for selecting probiotic bacteria as natural antagonists against pathogenic microorganisms [23]. Antimicrobial susceptibility tests revealed that the lyophilized CFS of L. brevis DPL5 had a strain-specific inhibitory effect on S. aureus cell growth. The MIC was in the range of 10 mg·mL−1 to 20 mg·mL−1 with an estimated probability (IBM SPSS Software) for MIC99 of 29.9 mg·mL−1 (Table 3). However, MIC had mostly a bacteriostatic (BS) effect on the tested nasal strains. The bactericidal concertation (BC) was dose dependent. For only 20% of the strains, BC was equivalent to MIC, while for the majority of strains, it reached up to 80 mg·mL−1. One-way ANOVA again showed no significant differences between MRSA and MSSA strains (p > 0.05), so subsequent analyses consider the two groups together. Based on the PROBIT analysis, the BC99 was calculated as 69 mg·mL−1. L. brevis, as a member of the genus Lacobacillus, is known for its potential to inhibit S. aureus [76,77], along with other species, such as L. fermentum, L. plantarum, L. acidophilus, L. casei L. rhamnosus and others [31,75,78,79]. In most cases, LAB are found to compete for colonization of the epithelial cells and control the overgrowth of pathogens [80], and only a few of them study the effect of extracellular extracts. Such antimicrobial activity for L. brevis is often a result of the production of organic acids (lactic and acetic acid) and/or bacteriocins, which inhibit the growth of Gram-positive pathogens [23,81]. Therefore, the demonstrated bactericidal properties of the LCFS of DPL5 highlight its probiotic potential.
The biofilm formation of S. aureus is considered a serious problem for public health and causes an increase in antibiotic resistance in chronic diseases and wound infection [82]. Currently, probiotic LAB metabolites are being evaluated along with natural compounds as an alternative to chemical agents to eradicate biofilm [22,83]. We investigated whether the LCFS from DPL5 facilitates the eradication of S. aureus biofilms. When strains were simultaneously treated with LCFS at 37 °C for 48 h, a concentration of 20 mg·mL−1 or higher led to a complete reduction in biofilm formation (p < 0.0001). The results are presented in Figure 5. The effect of lower concentrations was dose dependent, where 10 mg·mL−1 fully suppressed the biofilm formation in 32% of the strains (p < 0.0001) and significantly reduced the OD in the other 68% (p < 0.001). Concentrations of the LCFS as low as 5 mg·mL−1 significantly decreased the strength of the biofilms (p < 0.05) and were able to turn strong biofilms into weak ones. These results suggest that DPL5 LCFS effectively interferes with the biofilm formation of S. aureus. The estimated probability values for biofilm inhibitory concentration (BIC99) are presented in Table 2. These results conform to those of previous studies conducted on other lactobacilli [31,71,79]. In the present research, the established activity of LCFS subjected to pH neutralization showed that the antimicrobial activity is not a result of the production of organic acids. It suggests that the studied L. brevis DPL5 probably produced active metabolites against S. aureus. Moreover, the measurements of cell density that were obtained in conjunction with the examination of biofilm formation demonstrated that lower concentrations, which were less than the BIC50 threshold, did not result in the death of the nasal strains of S. aureus. According to Melo and collaborators [31], this demonstrates that the death of the pathogen did not decrease biofilm development. Such a statement is supported by the findings that the CFS from lactobacilli significantly down-regulates the virulence genes in S. aureus and interferes in the process of cell-to-cell communication [75,84], which leads to the inhibition of biofilm formation even without causing cell death. Furthermore, it confirms the production of modulatory chemicals capable of interfering with the pathogen’s ability to create biofilms.

3.7. Genomic Exploration of L. brevis DPL5 through Whole Genome-Sequencing

The genome of L. brevis DPL5 comprises a single circular chromosome measuring 2,334,043 base pairs (bps) with a coverage of ×105 and a guanine–cytosine (GC) content of 46.26%. Additionally, the strain contains two mobilizable plasmids with sizes of 44,900 bp and 30,428 bp and GC content 41.10% and 45.88%, respectively. The complete genomic sequences have been deposited in the NCBI database under accession numbers CP144904 for the chromosome and CP144903 and CP144905 for the plasmids.
Multilocus Sequence Typing (MLST) is a widely used technique for identifying and typing LAB [85]. This method involves analyzing the sequences of multiple housekeeping genes to generate a distinct allelic profile for each microorganism. MLST is highly regarded for its accuracy and for producing results that can be easily compared and shared across different studies [86]. For the DPL5 genome, the analysis through PubMLST confirmed with 100% certainty that it belongs to the L. brevis species. Additionally, phylogenomic analysis via genome-to-genome comparisons in TYGS revealed that the DPL5 strain clusters with other representative L. brevis strains in the database (Figure 6). These findings further confirm the classification of DPL5 as a member of the L. brevis species.
The strain DPL5 exhibits a robust genomic profile that underscores its potential as a versatile and resilient probiotic candidate. Notably, the strain is equipped with 50 genes dedicated to stress response, highlighting its adaptability and potential to survive in harsh and fluctuating environments such as the gastrointestinal tract [87]. This arsenal of stress-related genes likely enhances its resilience against oxidative stress, osmotic pressure, and other hostile conditions, ensuring its viability and functionality in probiotic applications. Detailed analysis showed that the genome harbored genes with probiotic activity for acid and bile tolerance, temperature stress, adhesion, antioxidant activity, and immunomodulation [88] (Table 4).
Equally impressive is the presence of 112 genes associated with the metabolism of cofactors, vitamins, prosthetic groups, and pigments. These genes suggest that DPL5 can synthesize or metabolize a variety of essential micronutrients, contributing not only to its survival but also potentially to the nutritional enhancement of its host [89]. This capability could be particularly beneficial in promoting gut health, aiding in the recovery of vitamin deficiencies, and supporting the overall metabolic balance of the host organism.
The DPL5 strain’s extensive genetic repertoire, comprising 251 carbohydrate and 272 protein metabolism genes, underscores its ability to utilize diverse dietary substrates and efficiently support amino acid turnover. The 103 genes involved in the cell wall and capsule biosynthesis highlight structural robustness and possible immune-modulatory properties [90]. Furthermore, the strain features 85 genes for DNA metabolism and 84 for RNA metabolism, which is indicative of efficient genomic maintenance and transcriptional regulation as well as critical for adapting to environmental shifts. A detailed genome annotation table can be found in Supplementary Table S2.
The genome of the L. brevis strain DPL5 reveals the presence of a few antimicrobial resistance (AMR) genes, including those encoding beta-lactamase classes A and C. Additionally, most Lactobacillus species exhibit intrinsic resistance to aminoglycosides [91], which is a characteristic corroborated by our antimicrobial susceptibility analysis and the genome identification of AMR genes such as ykkC, ykkD, and aacC. Furthermore, a comprehensive genomic scan using the ABricate tool and the Virulence Factor Database (VFDB) revealed no virulence factors. The absence of virulence genes in the DPL5 strain is a critical factor supporting its safety and potential as a probiotic. Virulence genes are often associated with pathogenicity, posing risks to host health, especially in immunocompromised individuals [92]. The lack of such genes ensures that DPL5 does not contribute to infections or disrupt the host’s natural microbiota. This safety profile is essential for probiotics, which promote health without adverse effects. Therefore, the absence of virulence genes in DPL5 underscores its suitability for applications in food, health supplements, and therapeutic interventions.
CAZy analysis is crucial for new bacterial strains, providing insights into their metabolic potential and ecological roles. By identifying specific carbohydrate-active enzymes, researchers can predict the strain’s ability to degrade, utilize, or transform various polysaccharides, which is essential for understanding its functionality in different environments. This information is particularly valuable in designing probiotics or industrial applications where targeted carbohydrate metabolism is critical. Furthermore, CAZy profiling can help identify unique or novel enzymes, paving the way for biotechnological innovations in food, biofuel, and pharmaceutical industries [93].
The analysis of CAZy enzymes in the DPL5 strain reveals a diverse repertoire of carbohydrate-active enzymes, highlighting its potential for utilizing a wide range of carbohydrates. The strain exhibits enzymatic capabilities for breaking down complex polysaccharides, including xylan, starch, and chitin, suggesting its adaptability to plant- and animal-derived substrates [94]. It also shows activity related to sucrose metabolism, indicating its ability to process common dietary sugars. The presence of enzymes targeting beta-glucuronan, beta-glucan, and beta-galactan further underscores its versatility in degrading various beta-linked polysaccharides. Additionally, enzymes associated with the metabolism of exo-polysaccharides, alpha-glucan, arabinan, and alpha-galactan expand the strain’s carbohydrate-processing spectrum. These features enhance the strain’s resilience in diverse environments and suggest its potential applications in probiotics and prebiotic development, where efficient carbohydrate utilization is a key trait.
The AntiSMASH tool can identify a wide range of biosynthetic gene clusters (BGCs), including those responsible for producing bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by bacteria. The results of the DPL5 strain showed lanthipeptide class IV, the T3PKS region and the RiPP-like region, all of which are chromosome encoded (Figure 7).
Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) known for their antimicrobial properties. Class IV lanthipeptides are characterized by their unique structural features, including multiple lanthionine rings. Identifying this class in a strain suggests the ability to produce compounds that can inhibit the growth of competing microorganisms, which may confer a competitive advantage in various ecological niches. RiPPs encompass a broad category of natural products derived from ribosomal synthesis followed by enzymatic modifications. The detection of RiPP-like regions suggests the presence of additional pathways for producing bioactive peptides that could function as antimicrobials or signaling molecules [95,96]. These compounds often play critical roles in microbial interactions and defense mechanisms. The diversity of RiPP-like regions is notable with different LAB genera exhibiting varying capacities for producing these compounds. The analysis showed that 28 out of 55 genera contained at least one RiPP per genome, indicating a widespread ability to synthesize these bioactive peptides. Furthermore, the study revealed that a high percentage of these gene clusters were specific to certain genera or species, suggesting that niche adaptation plays a critical role in distributing these biosynthetic capabilities. For instance, 92.6% of the identified gene clusters were genus-specific, underscoring the potential for unique metabolite production tailored to specific ecological niches [97]. The presence of RiPP-like regions in LAB is particularly significant for probiotic applications. These compounds not only possess antimicrobial properties but also play roles in shaping the microbiota by inhibiting pathogens and supporting beneficial microbial populations.
Type III polyketide synthases (T3PKS) are versatile enzymes involved in the biosynthesis of polyketides, which are secondary metabolites with diverse biological activities, including antimicrobial, antifungal, and anticancer properties. The presence of T3PKS regions indicates that the strain can synthesize complex polyketide structures that may have significant pharmaceutical potential [97].

4. Conclusions

The observations of the presented in vitro study indicated that the L. brevis DPL5 strain can survive in the gastrointestinal tract and industrial processes such as fermentation and freeze-drying processes. The strain demonstrated a significant antagonistic effect against nasal strains of S. aureus. The lyophilized cell-free supernatant of this LAB markedly reduced biofilm formation at concentrations <BIC50 and exhibited pronounced antimicrobial action at elevated concentrations (20 mg·mL−1), leading to the complete eradication of biofilms. The results suggest the production of bioactive extracellular compounds that are capable of interfering with the pathogen’s ability to create biofilms. The study highlights the probiotic potential of L. brevis DPL5. Conducting further studies on the extracellular production of biologically active metabolites by the strain studied would create an opportunity to develop biological alternatives to chemical dosage forms for topical treatment and the prevention/treatment of infections caused by biofilm-forming strains of S. aureus. In addition, genome-wide sequencing and in silico analysis of the L. brevis DPL5 using Nanopore technology reveals that it contains over 50 genes associated with probiotic activity, which may play a crucial role in its survival within the gut environment. The genome also includes multiple biosynthetic gene clusters that exhibit antibacterial properties.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microorganisms13010160/s1, Table S1: Results of antimicrobial susceptibility test for the nasal S. aureus isolates, interpreted according to EUCAST, 2024; Table S2: Levilactobacillus brevis DPL5 genome anotation.

Author Contributions

Conceptualization, V.B., I.I. (Ivan Iliev) and D.M.; methodology, V.B., I.I. (Ivan Iliev) and D.M.; software, V.B.; validation, I.I. (Ivan Iliev); formal analysis, V.B., I.I. (Ivan Iliev), M.G., E.A.-K., L.I. and G.Y.; data curation, V.B.; writing—original draft preparation, V.B., D.M. and I.I. (Ivan Iliev); writing—review and editing, V.B., I.I. (Ivan Iliev), V.G., E.A.-K., M.G., L.I., M.M., I.I. (Iliya Iliev) and G.Y.; supervision, V.B. and I.I. (Ivan Iliev). All authors have read and agreed to the published version of the manuscript.

Funding

This study is financed by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project BG-RRP-2.004-0001-C01.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of University of Plovdiv (Approval No. 6/06.10.2022).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lyons, K.E.; Ryan, C.A.; Dempsey, E.M.; Ross, R.P.; Stanton, C. Breast Milk, a Source of Beneficial Microbes and Associated Benefits for Infant Health. Nutrients 2020, 12, 1039. [Google Scholar] [CrossRef]
  2. Asenova, A.; Hristova, H.; Ivanova, S.; Miteva, V.; Zhivkova, I.; Stefanova, K.; Moncheva, P.; Nedeva, T.; Urshev, Z.; Marinova-Yordanova, V.; et al. Identification and Characterization of Human Breast Milk and Infant Fecal Cultivable Lactobacilli Isolated in Bulgaria: A Pilot Study. Microorganisms 2024, 12, 1839. [Google Scholar] [CrossRef]
  3. Zhang, X.; Mushajiang, S.; Luo, B.; Tian, F.; Ni, Y.; Yan, W. The Composition and Concordance of Lactobacillus Populations of Infant Gut and the Corresponding Breast-Milk and Maternal Gut. Front. Microbiol. 2020, 11, 597911. [Google Scholar] [CrossRef]
  4. Shewale, R.N.; Sawale, P.D.; Khedkar, C.D.; Singh, A. Selection Criteria for Probiotics: A Review. Int. J. Probiotics Prebiotics 2014, 9, 17–22. [Google Scholar]
  5. Binda, S.; Hill, C.; Johansen, E.; Obis, D.; Pot, B.; Sanders, M.E.; Tremblay, A.; Ouwehand, A.C. Criteria to Qualify Microorganisms as “Probiotic” in Foods and Dietary Supplements. Front. Microbiol. 2020, 11, 1662. [Google Scholar] [CrossRef]
  6. Li, S.W.; Watanabe, K.; Hsu, C.C.; Chao, S.H.; Yang, Z.H.; Lin, Y.J.; Chen, C.C.; Cao, Y.M.; Huang, H.C.; Chang, C.H.; et al. Bacterial Composition and Diversity in Breast Milk Samples from Mothers Living in Taiwan and Mainland China. Front. Microbiol. 2017, 8, 965. [Google Scholar] [CrossRef]
  7. Piqué, N.; Berlanga, M.; Miñana-Galbis, D. Health Benefits of Heat-Killed (Tyndallized) Probiotics: An Overview. Int. J. Mol. Sci. 2019, 20, 2534. [Google Scholar] [CrossRef] [PubMed]
  8. Zimmermann, P.; Curtis, N. Breast Milk Microbiota: A Review of the Factors That Influence Composition. J. Infect. 2020, 81, 78–88. [Google Scholar] [CrossRef]
  9. Soto, A.; Martín, V.; Jiménez, E.; Mader, I.; Rodríguez, J.M.; Fernández, L. Lactobacilli and Bifidobacteria in Human Breast Milk: Influence of Antibiotherapy and Other Host and Clinical Factors. J. Pediatr. Gastroenterol. Nutr. 2014, 59, 78–88. [Google Scholar] [CrossRef] [PubMed]
  10. Łubiech, K.; Twarużek, M. Lactobacillus Bacteria in Breast Milk. Nutrients 2020, 12, 3783. [Google Scholar] [CrossRef]
  11. Ayivi, R.D.; Gyawali, R.; Krastanov, A.; Aljaloud, S.O.; Worku, M.; Tahergorabi, R.; da Silva, R.C.; Ibrahim, S.A. Lactic Acid Bacteria: Food Safety and Human Health Applications. Dairy 2020, 1, 202–232. [Google Scholar] [CrossRef]
  12. Daif, A.; Zer, Y.; Erinmez, M. Lactobacillus Species in Breast Milk: Do They Get Affected by Birth Style? Clin. Exp. Health Sci. 2022, 12, 390–395. [Google Scholar] [CrossRef]
  13. Rahmati-Joneidabad, M.; Alizadeh Behbahani, B.; Taki, M.; Hesarinejad, M.A.; Said Toker, O. Evaluation of the Probiotic, Anti-Microbial, Anti-Biofilm, and Safety Properties of Levilactobacillus Brevis Lb13H. LWT 2024, 207, 116636. [Google Scholar] [CrossRef]
  14. Lee, Y.S.; Yu, H.Y.; Kwon, M.; Lee, S.H.; Park, J.I.; Seo, J.; Kim, S.K. Probiotic Characteristics and Safety Assessment of Lacticaseibacillus Casei KGC1201 Isolated from Panax Ginseng. J. Microbiol. Biotechnol. 2023, 33, 519–526. [Google Scholar] [CrossRef]
  15. Park, E.J.; Chun, J.; Cha, C.J.; Park, W.S.; Jeon, C.O.; Bae, J.W. Bacterial Community Analysis during Fermentation of Ten Representative Kinds of Kimchi with Barcoded Pyrosequencing. Food Microbiol. 2012, 30, 197–204. [Google Scholar] [CrossRef]
  16. FAO/WHO. Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria-Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food; FAO: Rome, Italy, 2001. [Google Scholar]
  17. De Chiara, I.; Marasco, R.; Della Gala, M.; Fusco, A.; Donnarumma, G.; Muscariello, L. Probiotic Properties of Lactococcus Lactis Strains Isolated from Natural Whey Starter Cultures. Foods 2024, 13, 957. [Google Scholar] [CrossRef]
  18. Deng, Z.; Hou, K.; Zhao, J.; Wang, H. The Probiotic Properties of Lactic Acid Bacteria and Their Applications in Animal Husbandry. Curr. Microbiol. 2022, 79, 22. [Google Scholar] [CrossRef] [PubMed]
  19. Tuo, Y.; Song, X.; Song, Y.; Liu, W.; Tang, Y.; Gao, Y.; Jiang, S.; Qian, F.; Mu, G. Screening Probiotics from Lactobacillus Strains According to Their Abilities to Inhibit Pathogen Adhesion and Induction of Pro-Inflammatory Cytokine IL-8. J. Dairy Sci. 2018, 101, 4822–4829. [Google Scholar] [CrossRef]
  20. EFSA BIOHAZ Panel (Panel on Biological Hazards). Update of the List of QPS-Recommended Biological Agents Intentionally Added to Food or Feed as Notified to EFSA 4: Suitability of Taxonomic Units Notified to EFSA Until March 2016; EFSA J 14:E04522; EFSA: Parma, Italy, 2016. [Google Scholar]
  21. Kim, S.H.; Kim, W.J.; Kang, S.S. Inhibitory Effect of Bacteriocin-Producing Lactobacillus Brevis DF01 and Pediococcus Acidilactici K10 Isolated from Kimchi on Enteropathogenic Bacterial Adhesion. Food Biosci. 2019, 30, 100425. [Google Scholar] [CrossRef]
  22. Kim, N.N.; Kim, W.J.; Kang, S.S. Anti-Biofilm Effect of Crude Bacteriocin Derived from Lactobacillus Brevis DF01 on Escherichia Coli and Salmonella Typhimurium. Food Control 2019, 98, 274–280. [Google Scholar] [CrossRef]
  23. Hojjati, M.; Behabahani, B.A.; Falah, F. Aggregation, Adherence, Anti-Adhesion and Antagonistic Activity Properties Relating to Surface Charge of Probiotic Lactobacillus Brevis Gp104 against Staphylococcus Aureus. Microb. Pathog. 2020, 147, 104420. [Google Scholar] [CrossRef] [PubMed]
  24. Tong, S.Y.C.; Davis, J.S.; Eichenberger, E.; Holland, T.L.; Fowler, V.G. Staphylococcus Aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management. Clin. Microbiol. Rev. 2015, 28, 603–661. [Google Scholar] [CrossRef] [PubMed]
  25. Grundmann, H.; Aanensen, D.M.; Van Den Wijngaard, C.C.; Spratt, B.G.; Harmsen, D.; Friedrich, A.W.; Sabat, A.J.; Muilwijk, J.; Monen, J.; Tami, A.; et al. Geographic Distribution of Staphylococcus Aureus Causing Invasive Infections in Europe: A Molecular-Epidemiological Analysis. PLoS Med. 2010, 7, e1000215. [Google Scholar] [CrossRef] [PubMed]
  26. Chen, L.; Tang, Z.Y.; Cui, S.Y.; Ma, Z.B.; Deng, H.; Kong, W.L.; Yang, L.W.; Lin, C.; Xiong, W.G.; Zeng, Z.L. Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus Aureus from Various Veterinary Hospitals. Pathogens 2020, 9, 264. [Google Scholar] [CrossRef]
  27. Rushdy, A.A.; Gomaa, E.Z. Antimicrobial Compounds Produced by Probiotic Lactobacillus Brevis Isolated from Dairy Products. Ann. Microbiol. 2013, 63, 81–90. [Google Scholar] [CrossRef]
  28. Azam, M.; Srivastava, R.; Ahmed, T. Isolation and Characterization of Bacteriocin Producing Levilactobacillus Brevis Strain ABRIINW-K from Buffalo Dung. J. Pure Appl. Microbiol. 2024, 18, 2137–2146. [Google Scholar] [CrossRef]
  29. Bergey, D.H. Vol 3: The Firmicutes. In Bergey’s Manual of Systematic Bacteriology; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2009. [Google Scholar]
  30. Menconi, A.; Kallapura, G.; Latorre, J.D.; Morgan, M.J.; Pumford, N.R.; Hargis, B.M.; Tellez, G. Identification and Characterization of Lactic Acid Bacteria in a Commercial Probiotic Culture. Biosci. Microbiota Food Health 2014, 33, 25–30. [Google Scholar] [CrossRef] [PubMed]
  31. Melo, T.A.; Dos Santos, T.F.; De Almeida, M.E.; Junior, L.A.G.F.; Andrade, E.F.; Rezende, R.P.; Marques, L.M.; Romano, C.C. Inhibition of Staphylococcus Aureus Biofilm by Lactobacillus Isolated from Fine Cocoa. BMC Microbiol. 2016, 16, 1–9. [Google Scholar] [CrossRef]
  32. Sharma, P.; Tomar, S.K.; Sangwan, V.; Goswami, P.; Singh, R. Antibiotic Resistance of Lactobacillus Sp. Isolated from Commercial Probiotic Preparations. J. Food Saf. 2016, 36, 38–51. [Google Scholar] [CrossRef]
  33. Haryani, Y.; Halid, N.A.; Guat, G.S.; Nor-Khaizura, M.A.R.; Hatta, M.A.M.; Sabri, S.; Radu, S.; Hasan, H. High Prevalence of Multiple Antibiotic Resistance in Fermented Food-Associated Lactic Acid Bacteria in Malaysia. Food Control 2023, 147, 109558. [Google Scholar] [CrossRef]
  34. György, É.; Laslo, É.; Antal, M.; András, C.D. Antibiotic Resistance Pattern of the Allochthonous Bacteria Isolated from Commercially Available Spices. Food Sci. Nutr. 2021, 9, 4550–4560. [Google Scholar] [CrossRef]
  35. Čuvalová, A.; Kmeť, V. Inhibition of Staphylococcus Aureus Biofilm by Lactobacillus Supernatant and Plant Extracts. J. Food Nutr. Res. 2018, 57, 250. [Google Scholar]
  36. Elleuch, L.; Shaaban, M.; Smaoui, S.; Mellouli, L.; Karray-Rebai, I.; Fourati-Ben Fguira, L.; Shaaban, K.A.; Laatsch, H. Bioactive Secondary Metabolites from a New Terrestrial Streptomyces Sp. TN262. Appl. Biochem. Biotechnol. 2010, 162, 579–593. [Google Scholar] [CrossRef]
  37. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in Vitro Evaluating Antimicrobial Activity: A Review. J. Pharm. Anal. 2016, 6, 71–79. [Google Scholar] [CrossRef] [PubMed]
  38. Magaldi, S.; Mata-Essayag, S.; Hartung De Capriles, C.; Perez, C.; Colella, M.T.; Olaizola, C.; Ontiveros, Y. Well Diffusion for Antifungal Susceptibility Testing. Int. J. Infect. Dis. 2004, 8, 39–45. [Google Scholar] [CrossRef] [PubMed]
  39. CLSI. Methods for Diluition Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 9th ed.; CLSI Document M07-A9; Clinical Laboratory Standards Institute: Wayne, PA, USA, 2012. [Google Scholar]
  40. Tatusova, T.; Dicuccio, M.; Badretdin, A.; Chetvernin, V.; Nawrocki, E.P.; Zaslavsky, L.; Lomsadze, A.; Pruitt, K.D.; Borodovsky, M.; Ostell, J. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res. 2016, 44, 6614–6624. [Google Scholar] [CrossRef]
  41. Overbeek, R.; Olson, R.; Pusch, G.D.; Olsen, G.J.; Davis, J.J.; Disz, T.; Edwards, R.A.; Gerdes, S.; Parrello, B.; Shukla, M.; et al. The SEED and the Rapid Annotation of Microbial Genomes Using Subsystems Technology (RAST). Nucleic Acids Res. 2014, 42, D206–D214. [Google Scholar] [CrossRef]
  42. Kanehisa, M.; Sato, Y.; Morishima, K. BlastKOALA and GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome Sequences. J. Mol. Biol. 2016, 428, 726–731. [Google Scholar] [CrossRef] [PubMed]
  43. Alcock, B.P.; Raphenya, A.R.; Lau, T.T.Y.; Tsang, K.K.; Bouchard, M.; Edalatmand, A.; Huynh, W.; Nguyen, A.L.V.; Cheng, A.A.; Liu, S.; et al. CARD 2020: Antibiotic Resistome Surveillance with the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 2020, 48, D517–D525. [Google Scholar] [CrossRef] [PubMed]
  44. Doster, E.; Lakin, S.M.; Dean, C.J.; Wolfe, C.; Young, J.G.; Boucher, C.; Belk, K.E.; Noyes, N.R.; Morley, P.S. MEGARes 2.0: A Database for Classification of Antimicrobial Drug, Biocide and Metal Resistance Determinants in Metagenomic Sequence Data. Nucleic Acids Res. 2020, 48, D561–D569. [Google Scholar] [CrossRef] [PubMed]
  45. Liu, B.; Zheng, D.; Zhou, S.; Chen, L.; Yang, J. VFDB 2022: A General Classification Scheme for Bacterial Virulence Factors. Nucleic Acids Res 2022, 50, D912–D917. [Google Scholar] [CrossRef] [PubMed]
  46. Gänzle, M.G.; Follador, R. Metabolism of Oligosaccharides and Starch in Lactobacilli: A Review. Front. Microbiol. 2012, 3, 340. [Google Scholar] [CrossRef]
  47. Goh, Y.J.; Klaenhammer, T.R. Genetic Mechanisms of Prebiotic Oligosaccharide Metabolism in Probiotic Microbes. Annu. Rev. Food Sci. Technol. 2015, 6, 137–156. [Google Scholar] [CrossRef] [PubMed]
  48. Becerra, J.E.; Yebra, M.J.; Monedero, V. An L-Fucose Operon in the Probiotic Lactobacillus Rhamnosus GG Is Involved in Adaptation to Gastrointestinal Conditions. Appl. Environ. Microbiol. 2015, 81, 3880–3888. [Google Scholar] [CrossRef] [PubMed]
  49. Saier, M.H.; Ye, J.J.; Klinke, S.; Nino, E. Identification of an Anaerobically Induced Phosphoenolpyruvate-Dependent Fructose-Specific Phosphotransferase System and Evidence for the Embden-Meyerhof Glycolytic Pathway in the Heterofermentative Bacterium Lactobacillus Brevis. J. Bacteriol. 1996, 178, 314–316. [Google Scholar] [CrossRef]
  50. Ye, J.J.; Saier, M.H. Allosteric Regulation of the Glucose:H+ Symporter of Lactobacillus Brevis: Cooperative Binding of Glucose and HPr(Ser-P). J. Bacteriol. 1995, 177, 1900–1902. [Google Scholar] [CrossRef]
  51. Cui, S.; Zhao, J.; Liu, X.; Chen, Y.Q.; Zhang, H.; Chen, W. Maximum-Biomass Prediction of Homofermentative Lactobacillus. J. Biosci. Bioeng. 2016, 122, 52–57. [Google Scholar] [CrossRef]
  52. Glaasker, E.; Konings, W.N.; Poolman, B. Osmotic Regulation of Intracellular Solute Pools in Lactobacillus Plantarum. J. Bacteriol. 1996, 178, 575–582. [Google Scholar] [CrossRef] [PubMed]
  53. Sleator, R.D.; Hill, C. Bacterial Osmoadaptation: The Role of Osmolytes in Bacterial Stress and Virulence. FEMS Microbiol. Rev. 2002, 26, 49–71. [Google Scholar] [CrossRef] [PubMed]
  54. Dodoo, C.C.; Wang, J.; Basit, A.W.; Stapleton, P.; Gaisford, S. Targeted Delivery of Probiotics to Enhance Gastrointestinal Stability and Intestinal Colonisation. Int. J. Pharm. 2017, 530, 224–229. [Google Scholar] [CrossRef]
  55. Wang, C.; Cui, Y.; Qu, X. Mechanisms and Improvement of Acid Resistance in Lactic Acid Bacteria. Arch. Microbiol. 2018, 200, 195–201. [Google Scholar] [CrossRef] [PubMed]
  56. Zhu, H.; Hart, C.A.; Sales, D.; Roberts, N.B. Bacterial Killing in Gastric Juice—Effect of PH and Pepsin on Escherichia Coli and Helicobacter Pylori. J. Med. Microbiol. 2006, 55, 1265–1270. [Google Scholar] [CrossRef] [PubMed]
  57. Mättö, J.; Alakomi, H.L.; Vaari, A.; Virkajärvi, I.; Saarela, M. Influence of Processing Conditions on Bifidobacterium Animalis Subsp. Lactis Functionality with a Special Focus on Acid Tolerance and Factors Affecting It. Int. Dairy. J. 2006, 16, 1029–1037. [Google Scholar] [CrossRef]
  58. Sarbini, S.; Hung, P.; Hassan, S. Evaluation of Bacteria Isolated from the Gastrointestinal Tract of Termites (Coptotermes Curvignathus) as Potential Probiotics. J. Agric. Food Dev. 2019, 5, 63–70. [Google Scholar] [CrossRef]
  59. Fontana, A.; Falasconi, I.; Molinari, P.; Treu, L.; Basile, A.; Vezzi, A.; Campanaro, S.; Morelli, L. Genomic Comparison of Lactobacillus Helveticusstrains Highlights Probiotic Potential. Front. Microbiol. 2019, 10, 1380. [Google Scholar] [CrossRef]
  60. Panghal, A.; Jaglan, S.; Sindhu, N.; Anshid, V.; Sai Charan, M.V.; Surendran, V.; Chhikara, N. Microencapsulation for Delivery of Probiotic Bacteria. In Nanotechnology in the Life Sciences; Springer: Cham, Switzerland, 2019. [Google Scholar]
  61. Chuah, L.O.; Shamila-Syuhada, A.K.; Liong, M.T.; Rosma, A.; Thong, K.L.; Rusul, G. Physio-Chemical, Microbiological Properties of Tempoyak and Molecular Characterisation of Lactic Acid Bacteria Isolated from Tempoyak. Food Microbiol. 2016, 58, 95–104. [Google Scholar] [CrossRef] [PubMed]
  62. Nozari, S.; Faridvand, Y.; Etesami, A.; Ahmad Khan Beiki, M.; Miresmaeili Mazrakhondi, S.A.; Abdolalizadeh, J. Potential Anticancer Effects of Cell Wall Protein Fractions from Lactobacillus Paracasei on Human Intestinal Caco-2 Cell Line. Lett. Appl. Microbiol. 2019, 69, 148–154. [Google Scholar] [CrossRef]
  63. Anisimova, E.A.; Yarullina, D.R. Antibiotic Resistance of LACTOBACILLUS Strains. Curr. Microbiol. 2019, 76, 1407–1416. [Google Scholar] [CrossRef] [PubMed]
  64. Campedelli, I.; Mathur, H.; Salvetti, E.; Clarke, S.; Rea, M.C.; Torriani, S.; Ross, R.P.; Hill, C.; O’Toole, P.W. Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp. Appl. Environ. Microbiol. 2019, 85, e01738-18. [Google Scholar] [CrossRef]
  65. Anisimova, E.; Gorokhova, I.; Karimullina, G.; Yarullina, D. Alarming Antibiotic Resistance of Lactobacilli Isolated from Probiotic Preparations and Dietary Supplements. Antibiotics 2022, 11, 1557. [Google Scholar] [CrossRef] [PubMed]
  66. Rokon-Uz-Zaman, M.; Bushra, A.; Pospo, T.A.; Runa, M.A.; Tasnuva, S.; Parvin, M.S.; Islam, M.T. Detection of Antimicrobial Resistance Genes in Lactobacillus Spp. from Poultry Probiotic Products and Their Horizontal Transfer among Escherichia coli. Vet. Anim. Sci. 2023, 20, 100292. [Google Scholar] [CrossRef]
  67. Das, D.J.; Shankar, A.; Johnson, J.B.; Thomas, S. Critical Insights into Antibiotic Resistance Transferability in Probiotic Lactobacillus. Nutrition 2020, 69, 110567. [Google Scholar] [CrossRef] [PubMed]
  68. Sabdaningsih, A.; Cristianawati, O.; Sibero, M.T.; Nuryadi, H.; Radjasa, O.K.; Sabdono, A.; Trianto, A. Screening Antibacterial Agent from Crude Extract of Marine-Derived Fungi Associated with Soft Corals against MDR-Staphylococcus Haemolyticus. IOP Conf. Ser. Earth Environ. Sci. 2017, 55, 012026. [Google Scholar] [CrossRef]
  69. Sikorska, H.; Smoragiewicz, W. Role of Probiotics in the Prevention and Treatment of Meticillin-Resistant Staphylococcus Aureus Infections. Int. J. Antimicrob. Agents 2013, 42, 475–481. [Google Scholar] [CrossRef] [PubMed]
  70. Gaspar, C.; Donders, G.G.; Palmeira-de-Oliveira, R.; Queiroz, J.A.; Tomaz, C.; Martinez-de-Oliveira, J.; Palmeira-de-Oliveira, A. Bacteriocin Production of the Probiotic Lactobacillus Acidophilus KS400. AMB Express 2018, 8, 153. [Google Scholar] [CrossRef] [PubMed]
  71. Koohestani, M.; Moradi, M.; Tajik, H.; Badali, A. Effects of Cell-Free Supernatant of Lactobacillus Acidophilus LA5 and Lactobacillus Casei 431 against Planktonic Form and Biofilm of Staphylococcus Aureus. Vet. Res. Forum 2018, 9, 301–306. [Google Scholar] [CrossRef]
  72. Colautti, A.; Orecchia, E.; Comi, G.; Iacumin, L. Lactobacilli, a Weapon to Counteract Pathogens through the Inhibition of Their Virulence Factors. J. Bacteriol. 2022, 204, e0027222. [Google Scholar] [CrossRef]
  73. Fornitano, A.; Amendola, I.; Santos, S.; Silva, C.; Leao, M. Lactobacillus Rhamnosus versus Staphylococcus Aureus: Influence on Growth and Expression of Virulence Factors. J. Dent. Maxillofac. Res. 2019, 2, 29–33. [Google Scholar] [CrossRef]
  74. Yüksek, F.K.; Gümüş, D.; Turan, D.B.T.B.; Nakipoğlu, Y.; Adaletï, R.; Küçüker, A.M. Cell-Free Supernatants of Lactobacilli Inhibit Methicilin-Resistant Staphylococcus Aureus, Vancomycin-Resistant Enterococcus and Carbapenem-Resistant Klebsiella Strains. Ege Tıp Dergisi 2021, 60, 332–339. [Google Scholar] [CrossRef]
  75. Ramezani, M.; Zainodini, N.; Hakimi, H.; Zarandi, E.R.; Bagheri, V.; Bahramabadi, R.; Zare-Bidaki, M. Cell-Free Culture Supernatants of Lactobacilli Modify the Expression of Virulence Factors Genes in Staphylococcus Aureus. Jundishapur J. Microbiol. 2019, 12, e96806. [Google Scholar] [CrossRef]
  76. Fang, F.; Xu, J.; Li, Q.; Xia, X.; Du, G. Characterization of a Lactobacillus Brevis Strain with Potential Oral Probiotic Properties. BMC Microbiol. 2018, 18, 1–9. [Google Scholar] [CrossRef]
  77. Singh, V.; Ganger, S.; Patil, S. Characterization of Lactobacillus Brevis with Potential Probiotic Properties and Biofilm Inhibition against Pseudomonas Aeruginosa. Proceedings 2020, 66, 14. [Google Scholar] [CrossRef]
  78. Al Kassaa, I.; Hober, D.; Hamze, M.; Chihib, N.E.; Drider, D. Antiviral Potential of Lactic Acid Bacteria and Their Bacteriocins. Probiotics Antimicrob. Proteins 2014, 6, 177–185. [Google Scholar] [CrossRef]
  79. Saidi, N.; Saderi, H.; Owlia, P.; Soleimani, M. Anti-Biofilm Potential of Lactobacillus Casei and Lactobacillus Rhamnosus Cell-Free Supernatant Extracts against Staphylococcus Aureus. Adv. Biomed. Res. 2023, 12, 50. [Google Scholar] [CrossRef] [PubMed]
  80. Bnfaga, A.A.; Lee, K.W.; Than, L.T.L.; Amin-Nordin, S. Antimicrobial and Immunoregulatory Effects of Lactobacillus Delbrueckii 45E against Genitourinary Pathogens. J. Biomed. Sci. 2023, 30, 1–19. [Google Scholar] [CrossRef]
  81. Chi, H.; Holo, H. Synergistic Antimicrobial Activity Between the Broad Spectrum Bacteriocin Garvicin KS and Nisin, Farnesol and Polymyxin B Against Gram-Positive and Gram-Negative Bacteria. Curr. Microbiol. 2018, 75, 272–277. [Google Scholar] [CrossRef]
  82. Parastan, R.; Kargar, M.; Solhjoo, K.; Kafilzadeh, F. Staphylococcus Aureus Biofilms: Structures, Antibiotic Resistance, Inhibition, and Vaccines. Gene Rep. 2020, 20, 100739. [Google Scholar] [CrossRef]
  83. Okuda, K.I.; Zendo, T.; Sugimoto, S.; Iwase, T.; Tajima, A.; Yamada, S.; Sonomoto, K.; Mizunoe, Y. Effects of Bacteriocins on Methicillin-Resistant Staphylococcus Aureus Biofilm. Antimicrob. Agents Chemother. 2013, 57, 5572–5579. [Google Scholar] [CrossRef]
  84. Mao, Y.; Wang, Y.; Luo, X.; Chen, X.; Wang, G. Impact of Cell-Free Supernatant of Lactic Acid Bacteria on Staphylococcus Aureus Biofilm and Its Metabolites. Front. Vet. Sci. 2023, 10, 01184989. [Google Scholar] [CrossRef] [PubMed]
  85. Prete, R.; Long, S.L.; Joyce, S.A.; Corsetti, A. Genotypic and Phenotypic Characterization of Food-Associated Lactobacillus Plantarum Isolates for Potential Probiotic Activities. FEMS Microbiol. Lett. 2021, 367, fnaa076. [Google Scholar] [CrossRef]
  86. Xu, H.; Liu, W.; Zhang, W.; Yu, J.; Song, Y.; Menhe, B.; Zhang, H.; Sun, Z. Use of Multilocus Sequence Typing to Infer Genetic Diversity and Population Structure of Lactobacillus Plantarum Isolates from Different Sources. BMC Microbiol. 2015, 15, 241. [Google Scholar] [CrossRef]
  87. Abriouel, H.; Pérez Montoro, B.; Casimiro-Soriguer, C.S.; Pérez Pulido, A.J.; Knapp, C.W.; Caballero Gómez, N.; Castillo-Gutiérrez, S.; Estudillo-Martínez, M.D.; Gálvez, A.; Benomar, N. Insight into Potential Probiotic Markers Predicted in Lactobacillus Pentosus MP-10 Genome Sequence. Front. Microbiol. 2017, 8, 891. [Google Scholar] [CrossRef]
  88. Mollova, D.; Gozmanova, M.; Apostolova, E.; Yahubyan, G.; Iliev, I.; Baev, V. Illuminating the Genomic Landscape of Lactiplantibacillus Plantarum PU3—A Novel Probiotic Strain Isolated from Human Breast Milk, Explored through Nanopore Sequencing. Microorganisms 2023, 11, 2440. [Google Scholar] [CrossRef]
  89. Rodionov, D.A.; Arzamasov, A.A.; Khoroshkin, M.S.; Iablokov, S.N.; Leyn, S.A.; Peterson, S.N.; Novichkov, P.S.; Osterman, A.L. Micronutrient Requirements and Sharing Capabilities of the Human Gut Microbiome. Front. Microbiol. 2019, 10, 1316. [Google Scholar] [CrossRef]
  90. Albarracin, L.; Raya Tonetti, F.; Fukuyama, K.; Suda, Y.; Zhou, B.; Baillo, A.A.; Fadda, S.; Saavedra, L.; Kurata, S.; Hebert, E.M.; et al. Genomic Characterization of Lactiplantibacillus Plantarum Strains Possessing Differential Antiviral Immunomodulatory Activities. Bacteria 2022, 1, 136–160. [Google Scholar] [CrossRef]
  91. Abriouel, H.; Casado Muñoz, M.d.C.; Lavilla Lerma, L.; Pérez Montoro, B.; Bockelmann, W.; Pichner, R.; Kabisch, J.; Cho, G.S.; Franz, C.M.A.P.; Gálvez, A.; et al. New Insights in Antibiotic Resistance of Lactobacillus Species from Fermented Foods. Food Res. Int. 2015, 78, 465–481. [Google Scholar] [CrossRef]
  92. Onyango, S.O.; Juma, J.; De Paepe, K.; Van de Wiele, T. Oral and Gut Microbial Carbohydrate-Active Enzymes Landscape in Health and Disease. Front. Microbiol. 2021, 12, 653448. [Google Scholar] [CrossRef]
  93. Sanders, M.E.; Akkermans, L.M.A.; Haller, D.; Hammerman, C.; Heimbach, J.; Hörmannsperger, G.; Huys, G.; Levy, D.D.; Lutgendorff, F.; Mack, D.; et al. Safety Assessment of Probiotics for Human Use. Gut Microbes 2010, 1, 164–185. [Google Scholar] [CrossRef]
  94. Lacombe-Harvey, M.È.; Brzezinski, R.; Beaulieu, C. Chitinolytic Functions in Actinobacteria: Ecology, Enzymes, and Evolution. Appl. Microbiol. Biotechnol. 2018, 102, 7219–7230. [Google Scholar] [CrossRef]
  95. Travin, D.Y.; Bikmetov, D.; Severinov, K. Translation-Targeting RiPPs and Where to Find Them. Front. Genet. 2020, 11, 226. [Google Scholar] [CrossRef]
  96. Han, S.-W.; Won, H.-S. Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs). Biomolecules 2024, 14, 479. [Google Scholar] [CrossRef] [PubMed]
  97. Zhang, D.; Zhang, J.; Kalimuthu, S.; Liu, J.; Song, Z.M.; He, B.B.; Cai, P.; Zhong, Z.; Feng, C.; Neelakantan, P.; et al. A Systematically Biosynthetic Investigation of Lactic Acid Bacteria Reveals Diverse Antagonistic Bacteriocins That Potentially Shape the Human Microbiome. Microbiome 2023, 11, 91. [Google Scholar] [CrossRef]
Microorganisms 13 00160 g001
Figure 1. Optical density (600 nm) after culturing in the presence of different sugars in a Lactobacillus brevis DPL5.
Figure 1. Optical density (600 nm) after culturing in the presence of different sugars in a Lactobacillus brevis DPL5.
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Microorganisms 13 00160 g002
Figure 2. Effect of different concentrations of sodium chloride on the growth of Levilactobacillus brevis DPL5.
Figure 2. Effect of different concentrations of sodium chloride on the growth of Levilactobacillus brevis DPL5.
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Microorganisms 13 00160 g003
Figure 3. Survival of L. brevis DPL5 in different acidic conditions.
Figure 3. Survival of L. brevis DPL5 in different acidic conditions.
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Microorganisms 13 00160 g004
Figure 4. Antagonistic activity against nasal Staphylococcus aureus strain: Plug diffusion test with 24 h (A), 48 h (B), and 72 h (C) L. brevis DPL5 cultivated on MRS agar. Agar well diffusion test showing the activity of L. brevis DPL5 cell-free supernatant (50 µL) after anaerobic cultivation in MRS broth for 24 h (D) and 48 h (E).
Figure 4. Antagonistic activity against nasal Staphylococcus aureus strain: Plug diffusion test with 24 h (A), 48 h (B), and 72 h (C) L. brevis DPL5 cultivated on MRS agar. Agar well diffusion test showing the activity of L. brevis DPL5 cell-free supernatant (50 µL) after anaerobic cultivation in MRS broth for 24 h (D) and 48 h (E).
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Microorganisms 13 00160 g005
Figure 5. Effect of different concentrations of lyophilized L. brevis DPL5 cell-free supernatant on the intensity of biofilm formation in nasal S. aureus strains.
Figure 5. Effect of different concentrations of lyophilized L. brevis DPL5 cell-free supernatant on the intensity of biofilm formation in nasal S. aureus strains.
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Microorganisms 13 00160 g006
Figure 6. Confirmation of the L. brevis DPL5 via genome-to-genome comparisons in TYGS.
Figure 6. Confirmation of the L. brevis DPL5 via genome-to-genome comparisons in TYGS.
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Microorganisms 13 00160 g007
Figure 7. Genomic maps of the clusters of the lanthipeptide class IV, T3PKS region, and RiPP-like region of the L. brevis DPL5.
Figure 7. Genomic maps of the clusters of the lanthipeptide class IV, T3PKS region, and RiPP-like region of the L. brevis DPL5.
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Table 1. Bile salt resistance of L. brevis DPL5, expressed as mean ± SD.
Table 1. Bile salt resistance of L. brevis DPL5, expressed as mean ± SD.
MediumBile Salts Concentration (%)
0.3%1%3%
MRS72 ± 0.3141 ± 0.236 ± 0.34
MRS with 7% lactose84 ± 05152 ± 0.2410 ± 0.13
Note: Growth of strain DPL5 in MRS or MRS with lactose broth was determined by measuring the absorbance at a wavelength of 600 nm after incubation at 37 °C. Bile resistance is expressed as a percentage of growth of the control (bile-free growth). Results were obtained from duplicate samples and were representative of 2 independent trials. Mean values with unlike superscript letters within a line differed significantly (p < 0.05).
Table 2. The antimicrobial susceptibility results of L. brevis DPL5 expressed as mean ± SD.
Table 2. The antimicrobial susceptibility results of L. brevis DPL5 expressed as mean ± SD.
Antibiotic CategoryName of AntibioticsDosage (µg)Diameter of
Inhibition Circle
(mm)		Drug
Sensitivity
AminoglycosidesAmikacin3011.32 ± 0.41R
TetracyclinesTetracycline3022.07 ± 1.15S
CephalosporinsCeftazidime1019.03 ± 0.68S
Cefazolin3022.51 ± 0.98S
Ceftriaxon309.61 ± 0.58R
Cefepime3021.45 ± 0.83S
QuinolonesCiprofloxacin521.5 ± 1.25S
β-Lactamase inhibitor complexAmpicillin/Sulbactam1026.08 ± 1.05S
Note: R indicates drug resistance; I indicates moderately sensitive; S indicates sensitive.
Table 3. Probability analysis for estimation of mean value and 95% confidence intervals for minimal biofilm inhibitory, minimal bacteriostatic, and bactericidal concentrations (mg·mL−1) for the 48 h cell-free L. brevis DPL5 lyophilized supernatant against nasal S. aureus strains based on the observed results.
Table 3. Probability analysis for estimation of mean value and 95% confidence intervals for minimal biofilm inhibitory, minimal bacteriostatic, and bactericidal concentrations (mg·mL−1) for the 48 h cell-free L. brevis DPL5 lyophilized supernatant against nasal S. aureus strains based on the observed results.
Biofilm Inhibitory ConcertationBacteriostatic
Concentration		Bactericidal
Concentration
BIC50BIC75BIC99MIC50MIC75MIC99BC50BC75BC99
Estimate probability9.84713.58929.90826.2331.6444.8828.30436.65669.048
Lower Bound8.29111.36621.74922.9127.8638.7624.11131.41552.738
Upper Bound11.83117.70453.75930.4537.4255.7133.22946.198117.907
Table 4. Probiotic gene markers identified in L. brevis DPL5.
Table 4. Probiotic gene markers identified in L. brevis DPL5.
Probiotic ActivityGenes
Acid and bile tolerancerpsS, pepF, kup, fabH, guaA, uvrA, dltA, dltC, dltD, pyk, recA, atpC, atpD, atpG, atpA, atpH, atpF, atpE, atpB,
Acid stress/bile resistance/temperaturegrpE, dnaJ, luxS, dnaK, eno, pgk. pgi, tpiA, gap, uvrA, nhaC
Adhesiontuf, lspA
AntioxidantmsrA, msrB, trxA, tpx, nrdH, mntH
Bile resistancerplD, rpsC, rplE, rplF, rpsE, argS, lpdA, glnA, pyrG
Cell wall formation or adhesionmurA
Cold stressrnr
ImmunomodulationdltB, dltC, dltD
Ionic and heavy metal stress resistancecorA
Oxidative stressmsrB
ProteasesclpX
Temperature stresshrcA, hslV, htpX
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Iliev, I.; Yahubyan, G.; Apostolova-Kuzova, E.; Gozmanova, M.; Mollova, D.; Iliev, I.; Ilieva, L.; Marhova, M.; Gochev, V.; Baev, V. Characterization and Probiotic Potential of Levilactobacillus brevis DPL5: A Novel Strain Isolated from Human Breast Milk with Antimicrobial Properties Against Biofilm-Forming Staphylococcus aureus. Microorganisms 2025, 13, 160. https://doi.org/10.3390/microorganisms13010160

AMA Style

Iliev I, Yahubyan G, Apostolova-Kuzova E, Gozmanova M, Mollova D, Iliev I, Ilieva L, Marhova M, Gochev V, Baev V. Characterization and Probiotic Potential of Levilactobacillus brevis DPL5: A Novel Strain Isolated from Human Breast Milk with Antimicrobial Properties Against Biofilm-Forming Staphylococcus aureus. Microorganisms. 2025; 13(1):160. https://doi.org/10.3390/microorganisms13010160

Chicago/Turabian Style

Iliev, Ivan, Galina Yahubyan, Elena Apostolova-Kuzova, Mariyana Gozmanova, Daniela Mollova, Iliya Iliev, Lena Ilieva, Mariana Marhova, Velizar Gochev, and Vesselin Baev. 2025. "Characterization and Probiotic Potential of Levilactobacillus brevis DPL5: A Novel Strain Isolated from Human Breast Milk with Antimicrobial Properties Against Biofilm-Forming Staphylococcus aureus" Microorganisms 13, no. 1: 160. https://doi.org/10.3390/microorganisms13010160

APA Style

Iliev, I., Yahubyan, G., Apostolova-Kuzova, E., Gozmanova, M., Mollova, D., Iliev, I., Ilieva, L., Marhova, M., Gochev, V., & Baev, V. (2025). Characterization and Probiotic Potential of Levilactobacillus brevis DPL5: A Novel Strain Isolated from Human Breast Milk with Antimicrobial Properties Against Biofilm-Forming Staphylococcus aureus. Microorganisms, 13(1), 160. https://doi.org/10.3390/microorganisms13010160

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