Complete genome sequencing and probiotic characterization of promising lactic acid bacterial strains isolated from dairy products in Egyptian markets

Abstract

Background

Probiotics refer to live bacteria that, when administered in a sufficiently, exert a beneficial influence on human health. Due to the probiotics' beneficial health advantages, dietary supplements are expanding rapidly as a self-care interest worldwide. It may be beneficial to administer probiotic strains resistant to antibiotics concurrently with an antibiotic treatment. Our study investigates nineteen dairy products collected from Egyptian markets, isolated, identified and underwent a characterization for probiotic features under demanding circumstances as NaCl, acid and bile salt environments. The antibiotic sensitivity test was performed later to the antimicrobial assessment against widespread both negative and positive gram-stained bacteria infecting humans, along with the antiviral evaluation against (SARS-CoV-2), the virus that has disturbed the world recently.

Results

Out of nineteen investigated isolates, five potential probiotic isolates were examined for probiotic characteristics. Our tested samples were of dairy origin (yogurt, cottage-cheese and sour milk) in Egypt, were identified as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Pediococcus acidilactici. These promising isolates had withstood stressful factors, such as NaCl, acid, bile salts, and the antimicrobial advance. The genomes were characterized for the physiology, safety, and efficacy of these isolates for probiotic qualities plus the presence of mobile genetic components and prophages that influence the genome's flexibility. They lack virulence factors and pathogenicity, rather than the lack of antibiotic resistance genes.

Conclusion

Three promising isolates underwent complete genome sequencing with high-throughput second generation technology followed by comprehensive bioinformatic analysis. The results showed that our isolates possess traits enabling resilience to antimicrobial effects and stress factors that might cause problems in the human gut. Several trustworthy genomic analysis methods were used to confirm and provide detailed illustrations of all traits. Genomic analyses confirmed the presence of stable genomes due to including mobile genetic components such as phages and CRISPR clusters, which validate their quality and safe usage for human health.

Introduction

Living bacteria classified as probiotics, can benefit human health when taken in the appropriate amounts [1]. The bacterial strain can be classified as a probiotic when satisfies the criteria for safety, functionality, and technological applicability. The safety criteria include human or animal origin; isolation from the alimentary tract of healthy individuals; confirmation of a safe use record; and absence of infectious disease potential [2]. The strain must meet the functional requirements of being resistant to the bacteriocins, acids, and bile salts produced by gut microbiota. Additionally, it must survive, metabolize, grow in the gut environment, and resist pathogenic colonization. Probiotics are technologically beneficial when their genomes are stable, resistant to bacteriophages, and end products are viable and durable throughout processing and storage [2]. Probiotics are recommended both before and after antimicrobial treatment to help restore the body's natural microbiota, support digestion, enhance nutrient and vitamin absorption, and alleviate food allergies [3]. Furthermore, probiotics are utilized to support the treatment of obesity, improve of lipid profiles and carbohydrate metabolism, and enhance host immunity [4]. Lactic acid bacteria (LAB) are a particularly prevalent, widely dispersed, and frequent resident of the human gut epithelium among the probiotic microbiota under investigation. Dairy products are rich source of LAB like processed yogurt and cottage-cheese, which is a fresh, un-ripened cheese made from milk, has a mild, tangy flavor and a soft creamy texture and is a good source of protein and calcium. LAB are a diverse group of Gram-positive bacteria that play a crucial role in various aspects of human life, particularly in food production and human health. Along with the human health benefits of LAB like gut health and immunity improvements, they have importance in food production as fermentation, preservation, flavor and texture [5].Many LAB species are widely employed in important traditional and industrial fermented dairy products, particularly Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Pediococcus acidilactici [6]. L. delbrueckii is a gram-positive, non-sporulating, cocco-bacilli or rod-shaped bacteria with a typical GC genome content of less than 50% [7]. Streptococcus thermophilus is gram-positive, non-motile and non-endospore-forming small, long chains of cocci that withstand elevated temperatures with a GC content less than 50% [8]. Pediococcus acidilactici is gram-positive paired or tetrad cocci, and can grow in a wide range of pH, temperature, and osmotic pressure [9]. These LAB were utilized as starter cultures in the production of various fermented dairy products, including cheese and yoghurt [10], and have been generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) [11]. Moreover, because it is dairy-derived, making its recipes for consumption would be easier.

Our study aims to investigate these local dairy isolates for their probiotic category through biochemical, stress endurance and molecular analyses which is further crowned by complete sequencing. Whole genome sequencing (WGS) of these isolates will serve as the foundation for in vitro safety evaluations, which may be verified by in vivo studies for pharmaceuticals intended for human use. WGS sheds light on the species' biology and helps identify genes unique to a particular strain, as well as its transcripts, probiotic attributes, and antimicrobial properties.

Methodology

Sampling and isolation

Nineteen milk-product samples (milk, sour milk, yogurt, and cottage cheese) were gathered from different Egyptian markets. We obtained 50 gm from each sample as 5 samples yogurt, 5 samples sour milk, 5 samples cottage-cheese and 4 samples raw milk. After packing and marking, the collected samples were immediately transferred in polyethylene bags on ice to the Main Chemical War laboratory near Nasr City in Cairo, Egypt [12]. Samples were homogenized by stomacher mixer in 10 mL buffered peptone water, incubated for 18 h at 37°C then, 1mL of the non-selective buffered peptone suspension was added to the de Man-Rogosa-Sharpe MRS broth (Merck, Germany) as enrichment media and incubated for 24 h at 37°C. MRS agar (Merck, Germany) plates were inoculated by 0.1ml grown samples and incubated at 37°C for 48h anaerobically (anaerobic jar and gas pack) [13].

Phenotypic characterization

To perform phenotypic characterization (morphological and biochemical), sub-culturing of single clear positive colonies was done on MRS agar plates three successive times for purification. Colonial morphology (colony shape, color, margin and opacity); gram staining and microscopic examination were inspected for each isolated culture [14]. Biochemical confirmation were done using the VITEK® 2C automated platform (bioMérieux, France) and the recommended ANC cards per the manufacturer's guidance [15].

Characterization for probiotic criteria

Confirmed gram-positive isolates were estimated for tolerance to NaCl, acids, bile salt, antibiotics and capability to suppress selected pathogens. According to the recommended procedure M7-A7-CLSI of Chemical Laboratory Standard Institute [16], each isolate's inoculum was made in tubes with 5 ml of broth media and incubated for 24h at 37°C. The inoculum's turbidity was sustained at 0.5 McFarland with 10⁷−10⁸ CFU/ml.

Tolerance to sodium chloride (NaCl)

NaCl tolerance was determined according to Uymaz Tezel et al. [17] with some modifications. Modified MRS broth (Merck, Germany) with different NaCl concentrations (0%, 3%, 4%, 5%, 6%, and 7%) (w/v) was applied. To the freshly prepared overnight culture, 1% (v/v) isolate was added into modified MRS and incubated anaerobically at 37ºC for (2–5) h intervals. The pure MRS broth acted as a control group, and the trial was performed in tripartite. At 600 nm, the absorbance was determined following two and five hours.

Acid and bile tolerance

As illustrated by Shaikh & Shah [18], isolates were investigated for viability an acidic medium. Overnight (1.3x 10⁹ CFU/ml) cultures in MRS broth were spun down at 5000 rpm for 10 min. After re-suspension of pellets in the PBS buffer which was modified at pH (3, 4, 5, 6) and pH 7 (control) and incubation at 37°C for 2h and 5h, 1% from each trial was added to new MRS broth (pH 7) and incubated for 24h at 37°C in tripartite pattern. The growth was measured at OD₆₀₀. Isolates with more than 50% resistance at pH 4, were designated acid-tolerant ones.

Each isolate was evaluated for tolerance to bile salt according the Vinderola and Reinheimer's approach [19]. Simply 200 µl from each isolated inoculum suspension (10⁷−10⁸ CFU/ml) were inoculated into 1000 µl of MRS broth with different doses of bile salt (0.1, 0.2, 0.3, 0.4 and 0.5% w/v) plus control MRS broth without bile salt and incubated for 2h and 5h at 37 C. Following incubation, the OD (optical density) was measured at 600 nm for comparison with the control. Isolates with more than 50% resistance at 0.3% bile concentration were designated bile-tolerant culture. The percent of tolerance was estimated in both circumstances (acid pH / bile values) as:

$${\%}\,\text{R}\text{e}\text{s}\text{i}\text{s}\text{t}\text{a}\text{n}\text{c}\text{e}=\frac{Increment\:of\:OD\:in\:MRS\:broth\:with\:bile\:salt/pH3,\:\text{4,5},\:6\:}{Increment\:of\:OD\:in\:MRS\:broth\:with\:bile\:salt/pH\:7}\times\:100$$

Antibiotics susceptibility

Using the disc diffusion technique, antibiotic sensitivity for each isolate was evaluated on Mueller-Hinton agar (MHA) [20]. The turbidity of the nocturnal tested inoculums (10⁷−10⁸ CFU/ml) was adjusted to 0.5 McFarland. MHA with methylene blue (0.5 g/ml) supplemented by 5% defibrinated horse blood and was inoculated by each isolate. BD BBL™ Sensi-Disc™ antibiotic susceptibility discs used in the test were (Ampicillin, Azithromycin, Ceftriaxone, Chloramphenicol, Ciprofloxacin, Clarithromycin, Erythromycin, Gentamicin, Nalidixic acid, Neomycin, Penicillin, Streptomycin, Tetracycline, Trimethoprim and Sulfamethizole). All discs were located on these streaked cultures by sterilized forceps, tested dishes were refrigerated for 2h and then incubated for 20h at 35°C. The zone of inhibition (ZOI) was measured according to the Laboratory and Clinical Standard Institute's guidelines [21].

Antibacterial activity

Antibacterial activity of all isolates against gram-negative and gram-positive pathogens (S. aureus, S. typhimurium, K. pneumonia, E. coli, E. fecalis, L. monocytogens) was assessed on MHA plates using the standard well diffusion procedure [22]. These tested pathogenic bacteria were obtained from the biobank of Main Chemical Warfare Laboratory. Each MHA plate was spread by 100 µl of pathogen inoculum (10⁷−10⁸ CFU/ml). Wells in each infected culture were inoculated by 30 µl cell-free solution obtained by centrifugation of 24h grown inoculum in MRS broth at 4000 rpm for 10 min. The negative control used was MRS broth, while the positive control was ciprofloxacin (30 ug/ml). To ensure that the suppression is not caused by lactic acid instead of the tested isolate, our supernatant broths were adjusted to pH 6.5. All dishes were incubated for 24h at 37^°C and the inhibition diameter was evaluated by automated colony counter Sphere Flash (IUL, Barcelona). Antibacterial activity was stated for isolates with ZOI measured 10 mm or more.

Antiviral activity

Cytotoxicity and plaque-reduction tests were conducted against the SARS-CoV-2 (COVID-19) virus from our biobank to evaluate the antiviral effect of promising isolates. The cytotoxicity effect of tested isolates against viability and proliferation of Vero E6 cells was done by the 3-(4,5-dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide (MTT) method in accord to Mosmann instructions [23] alongside slight alteration [24]. After preparing the stock concentration of the isolates in 10% DMSO, a serial dilution was done in Dulbecco’s Modified Eagle’s Medium (DMEM). Briefly, 100 µl of cell suspension with (3×10⁵ cells/ml) concentration were seeded and incubated for 24h at 37°C with 5% CO₂. Isolate dilutions were inoculated after 24h in triplicate. Following 24h, the media was aspirated, and cell lines were washed with PBS (phosphate buffer saline) 3 times to be inoculated by 20 µl MTT (5 mg/ml) then incubated for 4h at 37°C followed by medium aspiration. The crystalized formazan formed was solubilized by 200 µl ‎acidic isopropanol (0.04M HCl in pure isopropanol). Formazan solutions' absorbance was obtained using a compatible scanner at wavelength 540 nm, with 620 nm as a standard wavelength. The equation used to calculate the proportion of cytotoxicity versus untreated cells is as follows: [25, 26].

$${\%}\:\text{C}\text{y}\text{t}\text{o}\text{t}\text{o}\text{x}\text{i}\text{c}\text{i}\text{t}\text{y}=\frac{(Absorbance\:of\:untreated\:cells\:-Absorbance\:of\:treated\:cells)}{Absorbance\:of\:untreated\:cells}\times\:100$$

Calculating the cell viability reduced by 50% through plotting the sample concentration versus cytotoxicity percent is called (CC₅₀) [27].

The plaque reduction assay was done by seeding a six-well plate by Vero E6 cells (1×10⁶ cells/ml) at 37°C for 24h in 5% CO₂. 100 µl of the isolates’ safe dilutions (10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷) were mixed by (8×10⁴ PFU/well) plaque-forming unit of diluted COVID-19 then incubated at 37°C for 1h to be inoculated to the cells after aspirating the seeding medium. 1h incubation for virus adsorption was continued by adding 3 ml of supplemented DMEM (with 2% agarose). Solidified Plates were incubated for 3 days at 37°C (till viral plaques formed). After adding 10% formalin for 2h, 0.1% crystal violet was applied before washing. Positive control was assigned for wells infected with untreated virus plus a negative control well without virus. Lastly, the reduction percent in formed plaques compared to control ones was calculated using the following equation after counting the plaques: [25, 26].

$${\%}\:Reduction=\frac{(untreated\:viral\:count-treated\:viral\:count)}{untreated\:viral\:count}\times\:100$$

Using GraphPad Prism® (GraphPad Software Inc., San Diego, CA, USA) version 8, a nonlinear regression study was performed on the percent reduction data and the concentration values that corresponded to them to generate sigmoidal dose-response curves, by which the estimated concentration that would reduce the virus count by 50% (IC50) was derived [27]. The selectivity indices that measure the window between the cytotoxicity and antiviral activity [28] of the tested isolates were calculated according to the following equation: [25, 26].

$$\:Selectivity\:index=\frac{CC50\:of\:isolate\:on\:control\:Vero\:E6\:cells}{\text{I}\text{C}50\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{i}\text{s}\text{o}\text{l}\text{a}\text{t}\text{e}\:\text{o}\text{n}\:\text{i}\text{n}\text{f}\text{e}\text{c}\text{t}\text{e}\text{d}\:\text{V}\text{e}\text{r}\text{o}\:\text{E}6\:\text{c}\text{e}\text{l}\text{l}\text{s}}$$

DNA isolation

Using PureLink™ Genomic DNA purification kit (Thermo Fisher Scientific Inc, USA), genomic DNA extraction was done from the isolates’ broth. Per the manufacturer's guidelines, up to (2×10⁹) colonies were harvested after centrifugation and solubilized by Enzymatic Genomic Digestion Buffer. After adding Proteinase K, incubation at 55°C till cell lysis was done (30 min to 4h). After that, RNase A was added and mixed properly after incubation at room temperature for 2 minutes with Genomic Lysis/Binding Buffer. The whole content was mixed well with 100% ethanol and then washed twice by buffer AW1 and AW2 respectively. The eluted DNA was quantified for its concentration and purity by a previously calibrated Qubit™ 2.0 fluorometer instrument (Invitrogen, United States) with DNA HS assay kit and Nanodrop 8000 (Thermo Fisher Scientific, Waltham, MA, USA) as directed by the manufacturer, then kept at −20°C till used.

Genome sequencing

DNA libraries were prepared using the Nextera-XT DNA Library Prep kit (Illumina, San Diego, CA) following the manufacturer's guidelines with an initial DNA concentration of 1 ng/µl. By adding adapters of (i7) and (i5) indexes, Nextera transposome was used to fragment and tagment gDNA of tested samples. Amplified libraries were purified using single-sided Agencourt AMPure XP beads, normalized and diluted. Based on sample complexity, more than three samples might be pooled by run and then sequenced with the Illumina MiSeq 300-cycle cartridge kit for cluster generation on the Miseq Platform (Illumina, San Diego, CA, United States) at Genetics Research Unit, Main Chemical Warfare Laboratories, Ministry of Defense, Egypt.

Genome features, assembly and annotation

After the run had finished, primary analysis of the sequencing run parameters was done on board. FastQC (version 0.12.1) was applied to the raw reads [29]. Assembly quality study was processed by the comprehensive genome analyses tool at the PATRIC (PathoSystems Resource Integration Center) platform [30]. The obtained reads from previous analysis using FastQC and QUAST with good quality were annotated using RASTtk (Rapid Annotation using Subsystem Technology tool kit) [31].

Phylogenomic analysis

To identify the representative genomes and closest reference, Mash/MinHash was used [32]. From these genomes, PGFams (PATRIC global protein families) were chosen to ascertain the phylogenetic position [33]. These families' protein sequences were aligned using MUSCLE (Multiple Sequence Alignment) [34], and each sequence's nucleotides were allocated to the protein alignments. After concatenating the combined set of nucleotide and amino acid alignments into a data matrix, RaxML (Randomized Axelerated Maximum Likelihood) [35] was utilized to quickly bootstrap this matrix for analysis [36].

Screening of functional genes and genome stability

The cluster genes correlated to the probiotic properties were detected using (antiSMASH 7.0) [37]. The antibiotic resistance genes were predicted by ARTS 2.0 (Antibiotic Resistant Target Seeker) [38], AMR genes discovery tool based on k-mer in PATRIC and ResFinder software (version 4.5.0) [39]. The Bacteriocin Database BAGEL4 was applied to analyze the arrangement of the bacteriocin-encoding genes [40]. Presumed mobilome and phages, were searched under PhaBOX [41]. The CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR/Cas (CRISPR associated genes) were identified using the CRISPRCasFinder [42].

Statistical analysis

Standard deviation was estimated for each characterization experiment, which was done out in triplets. GraphPad Prism 6.0 was also used for statistical analysis. A p-value of less than 0.05 was deemed statistically significant.

Results

Identification of isolates and screening of the probiotic properties

Out of nineteen isolated samples, 15 samples gave identification on MRS selective media as white-smooth-mucoid rod-shaped colonies, small-white-smooth long chains of cocci and circular-white-smooth opaque colonies with entire margins and convex elevation existing in pairs respectively and gram-positive staining when has been applied for microscopic examination. Biochemical confirmation by the VITEK® 2C automated platform revealed five isolates as lactobacillus delbrueckii, Streptococcus thermophilus and Pediococcus acidilactici with excellent identification and was screened for the following probiotic criteria.

Tolerance to sodium chloride (NaCl)

Five LAB isolates were tested for their tolerance to different NaCl concentrations, out of which three tolerants only developed properly at doses of 3%, 4%, and 5%, while slow growth was noticed at concentrations of 6% and 7% NaCl. At 7% NaCl dose, the growth slowed greatly after both 2 and 5h of incubation (Table 1).

Table 1 Tolerance of isolated samples for NaCl concentrations at 2h and 5h

Acid tolerance

The three isolates that grew well at 5% NaCl concentration survived at low pH and showed resistance to acid stress. The resistant strains could survive at pH 4 (tolerance > 50%). After 5 h at pH 4, R1 had the highest tolerance (76.96%) and S6 had the lowest tolerance (68.86%) (Table 2). At pH 3, no tolerant strains were detected (resistance percentage < 50%).

Table 2 Acid tolerance of isolated samples at 2h and 5h

Bile tolerance

Inhibition was observed by increasing bile salt concentrations, but the three isolates tested continued to grow with increasing incubation time (resistance > 50%) but could not withstand bile salt concentrations of 0.5%. The highest resistance was reported for R1 (62.34%) and the lowest for G2 (50.66%), with 0.4% at 5h (Table 3). However, no isolates were resistant (percent resistance < 50%) in the 0.5% range. The challenges promising isolates face to survive these stressful conditions, which resemble those of the stomach and human intestine, are shown in Fig. 1a.

Table 3 Bile tolerance of isolated samples at 2h and 5h

Fig. 1

a The ability of isolated samples (G2- R1- S6- M3- M7) to survive against variable concentration of (pH – NaCl - Bile salt) after incubation in MRS broth for (2–5) h at 37° C. b Antibacterial activity of isolated samples against pathogenic bacteria

Antibiotics susceptibility

We investigated our trials for antibiotic resistance on MHA plates by disc diffusing technique and the results are presented in Table 4. Three isolates showed resistance to stress factors, and were highly sensitive to azithromycin, chloramphenicol, ciprofloxacin, erythromycin, clarithromycin, nalidixic acid and neomycin while sensitive to ampicillin, tetracycline, streptomycin, and gentamicin. Interestingly, these isolates share less sensitivity towards penicillin, trimethoprim, sulfamethizole and ceftriaxone. It would be advantageous if probiotic microorganisms could possess antibiotic resistance to survive antibiotic treatment in the gastrointestinal tract.

Table 4 Antibiotic susceptibility profile of isolated samples with MHA after 24 h

Antibacterial activity

Six pathogens were investigated to determine the antibacterial effects of the evaluated isolates (Fig. 1b). The bacterial cell-free fluids tested showed inhibition against the pathogenic strains examined. S. aureus was an extremely sensitive pathogen to all the tested isolates. Isolate G2 was the most potent isolate as in (Table 5).

Table 5 Zone of inhibition of Antibacterial activity against pathogenic bacteria

Antiviral activity

Since G2, S6, and R1 isolates demonstrated promising probiotic potential, further investigations were focused on these strains. The cytotoxicity of different concentrations of tested isolates (G2-R1-S6) on the viability of the Vero E6 cell line for 24 h, showed that 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷ were safe. These concentrations were subsequently tested for their antiviral effects against SARS-CoV-2.". The findings revealed an inhibition percentage as shown in Table 6. Isolate G2 was the active inhibitory one.

Table 6 Antiviral activity of effective antibacterial isolates against SARS-CoV-2

Genome sequencing

We obtained a complete genome sequence of three promising strains. A sequencing run was carried out on the Miseq platform for samples tested and identified during the study by the Nextera XT library preparation kit. The obtained data underwent a system analysis that resulted in 1350 k/mm2 total clusters, 90.8% a cluster PF, 96.5% Q30 (Read 1) and 97.5% Q30 (Read 2).

Genome features, assembly, annotation

The raw reads gave a good quality score on FastQC quality control checks on raw sequence data. According to the obtained data, sample no.1 coded by (G2) was assembled and resulting in 422 contigs with a genome length of 2,036,424 bp, With a typical G + C composition of 49.37%. The smallest sequence segment at 50% of the genome, which is known as N50 length, is 16,897 bp. The shortest number of contigs whose length sum produces N50 which defines the L50 count, is 33. Sample (G2) had been annotated with RASTtk (RAST tool kit) and given a distinct taxonomic classification as: cellular organisms > Bacteria > Terrabacteria > Firmicutes > Bacilli > Lactobacillales > Lactobacillaceae. This genome has 2,381 CDS (protein coding sequences), 61 tRNA (transfer RNA) genes, and 4 rRNA (ribosomal RNA) genes. The annotation involved 729 hypothetical proteins and 1,652 proteins having functional assignments out of which 559 EC numbers (proteins have Enzyme Commission), 463 GO assignments (proteins have Gene Ontology), and 388 proteins that were mapped to KEGG pathways and 2,232 proteins that belong to the PGFams (cross-genus protein families).

All these genomic features of our sequenced samples coded by (G2, R1 and S6) are listed in (Table 7). Samples coded by (G2 and S6) were assembled and resulted (1,016 and 19) contigs having (3,945,522 and 1,998,717) bp of genomic length of, with atypical GC content of (44.27 and 42.01) % respectively. The N50 length is (15,882 and 313,402) bp and the L50 count is (49 and 3) in the same respect. The annotation of sample (R1) revealed the taxonomy belongs to the family Streptococcaceae, while sample (S6) belongs to Lactobacillaceae. Both genomes have in order (4,902 and 1,963) CDS, (8 and 3) rRNA genes, and (89 and 15) tRNA genes. The annotation comprises (1,414 and 456) hypothetical proteins and (3,488 and 1,507) proteins have functional assignments divided into (1,225 and 547) EC proteins, (1,034 and 456) with GO assignments, and (866 and 387) proteins that had been mapped to KEGG pathways and (4,591 and 1,922) PGFams proteins.

Table 7 Assembly and annotation genomic features of the sequenced isolates

The distribution of the genome annotations is shown in a circular graph (Fig. 2a). From outer to inner rings, this comprises the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes, CDS with homology to known virulence factors, GC content and GC skew. The subsystem to which these genes belong is indicated by the colors of the CDS on the forward and reverse strands (Fig. 2a).

Fig. 2

a A circular graphical display of the contigs distribution through the genome map annotations. b A circular chart display of the subsystem (subsystems, genes). c A phylogenetic tree shows the most common strain our isolates belong to in the neighboring classification. d A graphical diagram showing the distribution of the secondary metabolites along the genome of R1 isolate

Subsystems are collections of proteins that work together to carry out a particular biological function or structure complex. Examination of subsystems unique to each genome is part of the PATRIC annotation process. (Fig. 2b) gives a summary of the subsystem values for these genomes. The most represented subsystem features are metabolism, protein processing, defense, stress response, energy, virulence, cellular processes, DNA processing, RNA processing, cell envelope, membrane transport, miscellaneous, regulation and cell signaling.

Phylogenetic tree analysis

Our strains G2, R1 and S6 showed maximum sequence similarity with Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophiles, and Pediococcus acidilactici, respectively. Based on the complete genomic sequence, a phylogenetic tree was created after concatenating the combined set of nucleotide and amino acid alignments into a data matrix, and RaxML was utilized to resolve this matrix to understand the phylogenetic relationship among LAB strains as shown in (Fig. 2c).

Bioinformatics analysis

Numerous annotated genes are homologous to known drug targets, transporters, and antibiotic-resistant genes. Antibiotic resistance phenotype is not always implied by the existence of AMR-related genes, even full ones, in a particular genome. This was confirmed through the ResFinder software (version 4.5.0), which proved that the samples were free of any resistant phenotypes. It is crucial to consider certain AMR pathways, particularly the existence or lack of SNP mutations that indicate resistance. Table 8 provides an overview of the AMR genes identified in this genome as the relevant AMR pathway.

Table 8 Specialty and AMR genes of the sequenced isolates

AntiSMASH enables quick discovery, annotation, and investigation of gene clusters involved in secondary metabolite production throughout the whole genome of bacteria. It identified the secondary metabolite regions (especially biosynthetic genes) that exist on different contigs along the genome of the analyzed isolates as shown in (Table 9).

Table 9 Biosynthetic secondary metabolites tracked by AntiSMASH software

The ARTS software confirmed all the resulting analyses obtained from AntiSMASH related to core/essential genes. Bacteriocin database BAGEL4 revealed the arrangement of the bacteriocin-encoding genes for G2 and R1 isolate as listed in Table 10, while no hits were found for S6 isolate.

Table 10 Bacteriocin-encoding genes localized on assembly contigs tracked by BAGEL4 software

Prophages and other insertion sequences which are named mobile genetic elements, were identified in the tested isolates by PhaBOX online tool. Two isolates were found to have phage sequences either virulent or temperate. Isolate G2 has six phage sequences out of which, one phage belongs to Peduoviridae family that matched Prevotella nigrescens as a suitable host. Isolate R1 showed thirteen phages, two of which belong to (Peduoviridae as isolate G2 and Ackermannviridae that matched Clostridioides difficile as suitable host). In Fig. 3a and b we found that the rest of the found phages have unknown families but matched a suitable host as shown in Tables 11 and 12. Isolate S6 doesn’t have any phage sequences.

Fig. 3

a A bar chart illustrating the abundance ratio of different phages among the tested samples G2 and R1. b An alignment of some phage sequences of G2 isolate to predict the suitable host

Table 11 Phage detected contigs in G2 isolate by PhaBOX

Table 12 Phage detected contigs in R1 isolate by PhaBOX

All isolates were searched for presumed CRISPR-Cas coding sequences. All three isolates showed these repeated sequences that contain one or more associated Cas-gene or consensus repeats and the spacer genes as shown in the following tables. They have (5, 11, 2) CRISPR sequences respectively, associated with (2, 7, 3) Cas in the same order and differentiated to (8, 25, 7) clusters in the arrange (G2, R1, S6) as in Tables 13, 14, and 15.

Table 13 CRISPR/Cas clusters detected in G2 isolate by CRISPRCasFinder software

Table 14 CRISPR/Cas clusters detected in R1 isolate by CRISPRCasFinder software

Table 15 CRISPR/Cas clusters detected in S6 isolate by CRISPRCasFinder software

Discussion

Probiotics are defined by the World Health Organization (WHO) as "living microbes that, when administered in adequate amounts, confer a health benefit on the host." Many food products available today contain live microorganisms added to promote general health benefits. Worldwide, probiotics are employed in a variety of products such as medicines, animal feed, and foods. In Egypt, all probiotics used in dietary supplements available on the medical market are imported. It became necessary to identify and describe a potential local probiotic strain for use in foods and medicines. The in vitro selection criteria for probiotics include bile and acid resistance, which enable these microorganisms to survive and thrive in the GIT (gastrointestinal tract) [43].

Nineteen samples from various Egyptian marketplaces, both natural and commercial, were collected and cultured on MRS agar. Among these, five isolates G2, R1, S6, M3 and M7 were confirmed with 99% excellent confirmation by VITEK system as Lactobacillus delbrueckii for G2, M3 and M7 isolates, Streptococcus thermophilus for R1 isolate and Pediococcus acidilactici for S6 isolate. These isolates subsequently underwent characterization for probiotic properties. Out of these five isolates, three isolates, G2, R1, S6 exhibited strong tolerance to 5% NaCl after 5h and survive well at pH 4 (with tolerance > 50%). Highest endurance was recognized in R1 (76.96%) while S6 showed the lowest (68.86%) at pH 4 after 5h (Table 2). However, none of the isolates exhibited tolerance at pH 3, as all had tolerance levels below 50%. Acid resilience is crucial for probiotic strains to survive the initial gastric shock in the stomach, where pH typically ranges between 2 and 3 [13].

Idoui [44] reported the persistence of L. plantarum BJ0021 at 3.0 pH and proved that, in contrast to isolated Lactobacillus from human gastrointestinal tracts, strains of L. fermentum, L. delbrueckii subsp. bulgaricus, and L. gasseri had greater acid endurance. Furthermore, acid-resistant isolates developed and persisted at a bile concentration of 0.4% for 5h. The highly resistant one was R1 (62.34%) and the lowest was G2 (50.66%) at 0.4% after 5h (Table 3). None of the isolates demonstrated tolerance at 0.5% bile, as all exhibited resistance below 50%. This aligned with the proof that the probiotic isolates were resistant to pH 3 and 0.3% bile [18]. As notice by Gilliland [45], lactobacilli isolated from animal intestines showed a greater resistance to bile salts when compared to those isolated from dairy products. showed. Streptococcus thermophilus and Pediococcus acidilactici under investigation are of dairy origin and are being assessed for their probiotic characteristics. Garriga et al. [46] revealed that chosen LAB strains are tolerant to 4% bile salt. LAB resistance varies due to the presence of bile salt hydrolase, an enzyme that conjugates bile to reduce its toxic effects [47].

One suitable criterion for selecting a probiotic culture is antibiotic sensitivity [48]. Using the agar disc diffusion method, the antibiotic sensitivity of all tested isolates was determined. Our selected isolates exhibited resistance to several antibiotics, consistent with findings by Botes et al. [49] who reported that L. casei was suppressed by various commercial antibiotics. Generally, Lactobacilli are susceptible to penicillin and β-lactamase targeting the cell wall but refractory to cephalosporins [50]. The bulk of LAB strains seem to be less inhibited by most inhibitors against nucleic acid synthesis. Lactobacilli, however, were often sensitive to low doses of many inhibitors of protein synthesis, like tetracycline and chloramphenicol, because of the existence of its resisting genes, which were occasionally detected in combination with another [51]. As antibiotic resistance among LAB is advantageous to survive in the GIT during antibiotic courses (REF), our resistant isolates to stress factors were highly sensitive to azithromycin, chloramphenicol, ciprofloxacin, erythromycin, clarithromycin, nalidixic acid and neomycin while sensitive to ampicillin, tetracycline, streptomycin, and gentamicin. Interestingly, these isolates share a common less sensitivity towards penicillin, trimethoprim, sulfamethizole and ceftriaxone.

Since probiotic bacteria regulate pathogenic species in the digestive system and boost immunity conferring a vital role to human health [52], antibacterial action is among probiotics' principal characteristics. Thus, the chosen isolates were examined against six pathogens as shown in Fig. 1b. All bacterial cell-free fluids tested showed inhibition against the pathogenic strains examined. S. aureus was an extremely sensitive pathogen to all the tested isolates. Isolate G2 was the most potent isolate. Tulumoglu and associates introduced the antibacterial efficacy of probiotics against S. aureus, P. aeruginosa, and E. coli [48]. PH values, struggle for nutrients, or the generation of compounds with a bacteriostatic or bactericidal effect, such as bacteriocin and bacteriocin-like compounds, may be related to probiotic strains' antibacterial activity [53], plus the antimicrobial metabolites [54], like acetic acid or lactic acid, carbon dioxide, diacetyl, aldehydes, hydrogen peroxide and other metabolites that serve as suppression substances for harmful bacteria [55].

Many previous studies stated that probiotics have good results in treating Covid-19 [56], even in high mutation rates [57]. As predicted, depending on the recent findings of probiotics effect in elimination of covid-19 virus [25], the antiviral activity toward SARS-CoV-2 after safe viability testing on Vero E6 cell-line for 24h was demonstrated by the successful antimicrobial isolates. The results in Table 6 revealed an inhibition percentage for G2 isolate which was the active inhibitory one.

The promising isolates G2, R1 and S6 exhibiting resistance against acid pH, bile salt and some antibiotics were picked for further research due to antibacterial and antiviral action. These isolates, derived from milk, were studied for probiotic qualities and investigated for safety and efficacy for human uses. All isolates were subjected to high-throughput complete genome next generation sequencing and analyzed for its functional characterization. Although this approach is still costly, WGS technology has become much less expensive over the past ten years, and this trend will continue when new systems are developed [58].

DNA next-generation sequencing has rapidly developed into an extensive technique for material identification and analysis. The WGS resulting data was analyzed using several pipelines, outlined in the methodology. This approach can be customized to provide the required taxonomic accuracy, for example, strain-scale microorganism identification [59]. Overall, high-throughput sequencing provided amazing information that summed up the significance of the strains that were evaluated. WGS was done for G2, R1 and S6 isolates with excellent parameters resulting from onboard primary analysis with 1350 k/mm2 total clusters, 90.8% a cluster PF and 97% total Q30, then the raw reads underwent further check and gave a good quality score on FastQC tool.

After assembly, all reads were annotated revealing 422 contigs with 2.04 Mbp genome length and 49.37% GC content for the G2 isolate, 1016 contigs with 3.95 Mbp genome length and 44.27% GC content for the R1 isolate, and 19 contigs with 1.99 Mbp genome length and 42.01% GC content for S6 isolate. Omic-based techniques and genetic sequencing can effectively identify probiotic-related genes, including those linked to metabolism, genomic adaptability, stability, antimicrobial tolerance, virulence, and safety In addition, in vivo, in vitro, and omic research are essential tools for assessing probiotic abilities, including tolerance to acidic environments, bile acids, antimicrobial substances, immuno-modulation, and adhesive mechanisms [60]. Probiotics benefit recipients' health by modifying their gut microbiota, therefore limiting pathogen colonization, regulating the host immunity response, reducing serum cholesterol, exhibiting antidiabetic, antihypertensive and antioxidant actions, and creating bacteriocins [61]. Phylogenetic analysis revealed that our isolates are closely related to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Pediococcus acidilactici. Phylogenetic trees were created after the combined set of nucleotide and protein alignments were placed into a concatenated matrix and analyzed by PATRIC and RaxML as a part of comprehensive genome analysis. All these data were deposited in the NCBI database.

The genome subsystem analysis, represented in Fig. 2b, includes protein processing, metabolism, stress response, defense, virulence, DNA processing, cellular processes, energy, RNA processing, membrane transport, cell envelope, miscellaneous, regulation and cell signaling. In our study, the genome was analyzed for the screening of antimicrobial tolerance, mobile genetic elements as prophages and the CRISPR/Cas system. The concern with antibiotic resistance is that pathogenic microorganisms may acquire antibiotic-resistant genes from the microbiota [62]. By online bioinformatics tools ResFinder and ARTS, the genome analysis showed that, no antibiotic resistance-related phenotypes or genotypes were found. LAB is known for their antibiotic sensitivity according to in vitro studies [63]. Most likely, the environment or stress that the bacteria were subjected to influences this finding. The resulting analysis of ARTS software related to core/essential genes confirmed all obtained from AntiSMASH. BAGEL4 tool revealed the existence of the bacteriocin-encoding genes for G2 and R1 isolate, while no hits were found for S6 isolate by these core/essential genes by ARTS, AntiSMASH and BAGEL4.

PhaBOX online tool discovered prophage sequences that were either virulent or temperate. Most identified phages belong to Peduoviridae and Ackermannviridae families that matched different suitable hosts as L. delbrueckii and Streptococcus thermophilus for both G2 and R1 strains, while no prophage sequences were found in S6 strain.

Prophages, on the other hand, contribute to the host's genome stability by modulating the individual's growth and survival in the GIT environment while also protecting the host cells from invading virulent bacteriophages and antimicrobial substances [64].

As shown in Tables 13, 14, and 15, our isolates contain CRISPR clusters in their genomes, along with Cas-genes and spacers. The existence of CRISPR clusters within a genome restricts the spread of antimicrobial resistance genes by impeding several mechanisms of horizontal transfer of genes [65]. The existence of efficient CRISPR slices provides a strain with a sequence-specific fighting barrier against phages, plasmids and insertion sequences. The CRISPR loci, with related Cas genes, also give the host strain the ability to combat itself from any invading extra-chromosomal genetic molecules [66]. This is a marker of our analyzed genome stability and a very minimal likelihood of the strain acquiring antimicrobial resistance genes, as resistant genes are generally transferred by mobile genetic elements.

Conclusion

In conclusion, five potential probiotic isolates out of nineteen investigated isolates were examined for probiotic characteristics. The promising samples, obtained from local dairy products (yogurt and sour milk) in Egypt, were confirmed as L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Pediococcus acidilactici. They demonstrated resilience to stressful environments, including NaCl, acid, bile salts, and the antimicrobial activity. The study suggests that probiotic strains resistant to antibiotics may have potential for co-administration during antibiotic treatments. Using an in-silico approach, the isolates were characterized for their physiology, safety, and efficacy. Its positive attributes are highlighted by the lack of virulence factors and pathogenicity in this study, which makes it a good choice for usage as a probiotic supplement. While the isolates lack genes conferring antibiotic resistance, the presence of mobile genetic elements and prophages may contribute to genome flexibility. R1 isolate showed the most promising probiotic properties and beneficial genetic features, followed by G2 isolate, while S6 isolate lacked some necessary genetic elements, resulting in a lower overall assessment. Further in vivo studies on animal and human clinical trials are needed to confirm the safety, efficacy, and health benefits of these promising isolates for probiotic use.