Introduction

Probiotics are known as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [1]. Presence of adequate scientific evidence of safety, viability, and efficacy should exist to consider strains as probiotics, and this distinguish them from strains with probiotic potential or potentially beneficial microbes [1]. The potential health benefits of probiotics include improving immune responses [2], eliminating gastrointestinal microbial pathogens [3], strengthening intestinal mucosal barrier [4], reducing cholesterol level in serum [5], and treating many diarrheal and gastrointestinal diseases [6, 7].

A growing evidence about probiotics to be useful in medicine such as reducing incidence of traveler’s diarrhea [8], reducing severity of rotavirus gastroenteritis [9], and remission from Clostridium difficile colitis [10] has been documented. Randomized controlled trials from human studies accumulate evidences for health effects of probiotics. However, it is plausible to distinguish such effects which are widely distributed among probiotics such as normalizing disturbed microbiota, exclusiveness of pathogens, colonization resistance, and prevention of gastrointestinal diseases [1] and those effects that are rare among probiotics. In this context, evidences for specific health benefits pertaining to specific probiotic strains are on the rise. For example, probiotics may reduce eczema-relative risk by 22% during late pregnancy [11] or reduce crying time in breastfed infants by average of 25 min per day [12]. Additionally, probiotics have been realized to impart beneficial effects at distant sites such as the airways, skin, brain, and heart [13].

Evidences for probiotics’ benefits were based on using diverse microbial species in experimental and clinical settings. However, Lactobacillus and Bifidobacterium strains received enormous interest as therapeutic agents. Clinical studies showed efficacy of L. rhamnosus GG and L. acidophilus as well as B. longum in treatment of colon cancer and colorectal cancer, respectively [14, 15]. Strains belonging to L. plantarum, L. reuteri, and L. brevis were found efficient in treatment of vaginal disorders [16, 17]. In addition to this, there are evidences for efficacy of Lactobacillus and Bifidobacterium strains in treatment of hypercholesterolemia [18], inhibition of Helicobacter pylori infection [19], treatment of oxaluria [20], treatment of gastrointestinal pathologies [21], survival in gastrointestinal tract [22], and treatment of acute diarrhea [23]. The growing scientific evidence of using these two genera prompted us to explore some beneficial characteristics of six strains belonging to the same genera in the current study.

The aforementioned findings highlight the significant role of probiotics in prevention and treatment of human diseases and to be the game changers in preventive medicine. Despite the numerous studies focused on health-promoting benefits of probiotics [24], boosting the immune system and preventing infections [25], their interaction with host [26], their role in balancing the human gut microbiome [27], and the improving remedy of metabolic diseases [28], the development of personalized treatments which is based on truly patient profile are still lacking and screening for the strains to be used for such therapy under in vitro and in vivo conditions is mandatory. With regard to predictive medicine approaches, probiotics could serve as a basis for a panel of biomarkers for recognition of immunity-related, and respiratory diseases or cancer based on their role in the gut-brain axis or the intestinal microbiota-human interactions [29]. Being of natural origin, well-documented characterization of the potent probiotic strains will contribute to the move from reactive to preventive, personalized, and predictive medicine. In this regard, strains of probiotic L. delbrueckii, L. casei, and B. animalis were proven to reduce cholesterol levels in blood serum and recover liver morphology of obese BALB/c mice as described by Bubnov et al. [30]. In the same study, probiotics were able to decrease body weight and beneficially module gut microbiota of the fat-diet-fed mice. These findings suggest likelihood of using such strains in prevention and treatment of metabolic syndromes. However, consideration regarding strain-dependent properties (e.g., resistance to biological fluids and antibiotics) of Lactobacillus and Bifidobacterium strains should be made prior to their applications in individualized treatment [31]. Since we had evidence about the antagonistic and antibiofilm potential of the six probiotics strains, used in this study, we initiated current investigation to reveal additional beneficial traits that can expand the strains’ applicability against microbial infections. This also will lay the ground for designing future animal studies to confirm their beneficial use.

The application of probiotics as therapy necessitates their safety and maintenance of host cell viability. According to the International Association of Probiotics and Prebiotics (ISAPP) consensus document, certain effects could be ascribed to probiotics as a general class based on well-designed trials, meta-analyses, and systematic reviews. In the same document, several bacterial species (e.g., L. acidophilus, L. plantarum, L. rhamnosus, B. longum, and B. bifidum), from which some strains have been investigated here, were mentioned to be accepted by Health Canada for their contribution in gut microbiota health [1]. Collectively, this implicates their safety for human use. Safety of probiotic bifidobacteria and lactobacilli has been extensively overviewed. Di Cerbo et al. [32] summarized clinical evidences that support the therapeutic efficacy of lactobacilli in the treatment of numerous pathological conditions based on selected papers from 1950 to 2015. Different approaches have been proposed to measure safety of probiotics. Of these, monitoring toxicity in vivo via evaluation of some parameters such as blood parameters, assessment of bacterial translocation, signs of intestinal inflammation, or alterations in body or splenic weight [33]. Additional methods include measuring infectivity in vitro or in animal models [33]. However, little is known about the safety of the strains’ supernatants after their growth in conventional media. This inspired our current work to assess the safety of cell-free preparations of six probiotics, belonging to Lactobacillus and Bifidobacterium spp., which demonstrated antibacterial and antibiofilm potential in our previous study against multidrug resistance Escherichia coli [34].

The use of probiotics as antiviral agents was reported [35, 36]. For example, B. adolescentis SPM0212 exerted antiviral activity against Hepatitis B virus [37] and B. adolescentis SPM1005 suppressed human papillomavirus–associated cervical cancer [38]. Bifidobacterium and Lactobacillus spp. proved to be antagonistic against many viruses such as herpes simplex virus, human influenza virus, and human immunodeficiency virus [39,40,41]. However, the efficacy of probiotics as antagonistic agents against some viruses was rarely investigated. Of these viruses are Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV). The former is the causative agent of Newcastle disease (ND) which is a highly infectious disease that has been reported globally and associated with multiple outbreaks in poultry [42]. On the other hand, IBDV is the causative agent of infectious bursal disease (IBD) which is highly contagious and acute in young chicken and turkeys [43, 44]. Currently, there is no effective treatment for NDV and IBDV, and protective vaccination is the only therapy adopted. This prompted us to assess the in vitro antiviral effect of probiotics against NDV and IBDV to gain insights about their possible use in further animal studies.

Genome mining is a powerful tool in unraveling the genetic determinants involved in host-microbe interactions. The increasing capabilities of DNA sequencing technologies enabled identifying majority of the genes in probiotic genomes [45, 46]. The approach has been utilized to advance the understanding of the probiotic traits such as host adhesion factors in VSL#3 multispecies marketed probiotic [45]. In addition, functional genomics elucidated genes of probiotic traits in Bacillus coagulans HS243 [46]. Furthermore, comparative genomic analysis of Enterococcus sp. genomes emphasized the genetic repertoire that confers probiotic features [47]. Genome mining analysis is utilized in current work to uncover the presence of genomically encoded adherence factors in the six probiotic strains that we investigated previously [34].

Our aim is to provide additional evidence for the probiotic traits, throughout evaluating the cytotoxicity and antiviral activity, of different probiotics belonging to Lactobacillus and Bifidobacterium spp. against Newcastle disease and infectious bursal disease viruses. Additionally, we will utilize genome mining tools to unravel the host adherence factors in these probiotics to emphasize their potential use in health promotion and prophylaxis.

Methods

Bacterial strains and growth conditions

Six strains of probiotics, obtained from Egyptian Microbial Culture Collection, Microbiological Resources Centre, Ain-Shams University, Egypt, were used in this work as shown in Table 1. Probiotic strains were grown on Man-Rogosa-Sharpe (MRS; Oxoid, Hampshire, England) agar for 24 h at 37 °C with 5% CO2. Single colony from each strain was transferred into MRS broth under the same incubation conditions for 24 h for the preparation of the cell-free spent medium (CFSM).

Table 1 Probiotic strains used in this study
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Preparation of the cell-free spent medium

The preparation of CFSM from each probiotic strain was performed as described previously [34, 48]. Briefly, overnight cultures of the six probiotic strains grown in MRS broth at 37 °C were diluted 1:100 with fresh medium and allowed to grow under same conditions to an optical density of 1.6 (~ 1 × 108 cells/mL), and the cells were then removed using centrifugation at 6000 xg at 4 °C for 10 min. The supernatant was filter-sterilized through 0.2-μm pore-size filter (Sigma Aldrich, Munch, Germany) and referred to as CFSM. The CFSM of all probiotic strains was stored at − 20 °C until use for cytotoxicity and antiviral assays.

Cytotoxicity assay for the CFSM of probiotics

The cytotoxicity assay was conducted using a Baby hamster kidney (BHK-A) cells (Sigma Aldrich) as described previously [49]. The BHK-A cells were grown in Dulbecco’s modified Eagle’s medium (DEME; Sigma scientific services, Cairo, Egypt) supplemented with 200 μM glutamine and 10% fetal calf serum and incubated at 37 °C under 5% CO2 for 24 h. The cells were inoculated at a density of 1 × 104 and allowed to grow until the cell has become confluent. Filtered CFSM at different dilutions (10%, 50%, 75%, and 100%) were added to the cell culture and incubated at the same conditions for 48 h. After incubation, the cells were fixed, washed, and stained with sulfo-rhodamine B stain. Excess stain was removed using acetic acid while the bound stain was recovered by Tris-EDTA buffer. The color intensity of the stain was measured in an ELISA reader (Sun Rise–TECAN, Clinilab Ltd., Egypt). The untreated cells were considered as a control. The surviving fraction of BHK cells in each treatment was calculated relative to the control.

Antiviral assay

Virus stock

Pure NDV and IBDV were implemented in this study and obtained kindly from The Virology Lab., Department of Microbiology, Faculty of Agriculture, Ain-Shams University, Egypt.

Cell line

Primary Chicken embryo fibroblast (CEF) cell culture (supplied by Virology Department, Animal Health Research Institute, Cairo, Egypt) was prepared using 9- to 10-day-old chicken embryos by warm trypsinization method [50]. Viability of the cells was determined by using “dye exclusion method,” as described before [50, 51]. Micro-titration of NDV and IBDV was performed using the same cell line as described previously [52].

Seeding of CEF cell culture

For antiviral assay, 100 μl of CEF (1 × 105 cells/mL), grown in Eagle’s minimal essential medium (MEM; GIBCO, Grand land, NY, USA) containing 10% inactivated calf serum and 100 μg/mL penicillin and streptomycin, were seeded in each well of the 96-well plates. The plates were then incubated at 37 °C for 72 h with 5% CO2. Cells in each plate were regularly monitored under an inverted microscope (HUND, Germany) until they reached to a confluence state of 80–90% [50, 51].

Antiviral activity

The antiviral activity was investigated as reported before [50, 51]. Briefly, to determine any cytotoxic potential of CFSM on CEF cells, 100 μl of 10% probiotics’ CFSM were added to confluent CEF cells grown in freshly prepared media, while CEFs grown only in Eagle’s minimal essential medium (EMEM) served as positive control. Additionally, DMSO (20%) and EMEM media was used as negative control. For antiviral assay, 50 μl from each probiotic CFSM, at 10% final concentration, was mixed with 50 μl ND and IBD virus (106 and 104 tissue culture infectious dose (TCID) 50, respectively). The test concentrations (100 μl total) were added to each well containing CEF cells. Afterwards, plates were kept at 37° C in 5% CO2. The development of cytopathic effects was observed every 24 h for 3 days. The reading of CPE was recorded, and the virus titer was calculated as described before [52].

Genome mining analysis for host adherence proteins

The whole genome sequences of the six probiotic strains were retrieved from the NCBI using the accession numbers listed in Table 1. The annotated genomes were screened for host adherence factors using the NCBI sequence set browser. The adherence factors were selected based on previous reports that defined the genetic determinants necessary for probiotic Lactobacillus and Bifidobacterium spp. to adhere to host tissues [45, 53]. The retrieved protein sequences of adherence factors in the probiotic strains were screened for similarity using online NCBI’s BLASTp tool. In addition, protein sequences of adherence factors were analyzed for similarity with translated nucleotide sequences from genomes of commercial probiotic strains using tBLASTn tool. For B. bifidum JCM 1254, the annotated protein sequences were not available on NCBI database and the genetic determinants encoding adherence proteins in the strain’s genome were identified based on sequence similarity with the closest reference genome. Genes encoding adherence factors in B. bifidum JCM 1254 genome were screened for sequence similarity with the reference genome in addition to a marketed probiotic B. animalis BB-12 strain using BLASTn tool. Genes encoding bile salt tolerance were identified using tBLASTn tool. Antibiotic resistance genes (ARGs) in the probiotic strains were analyzed against the comprehensive antibiotic resistance database (CARD).

Results

Cell-free spent media and cytotoxicity

The cytotoxic activities of four different dilutions of the cell-free preparations of the six probiotic strains were investigated using BHK cell line in vitro. The different preparations of CFSM exhibited very low toxicity as indicated by the high survival rate of BHK cells (surviving fraction ranged from 0.82 to 0.99) as compared to the control as shown in Table 2. Only, in case of the CFSM of L. helveticus, the cell viability was lower than the other strains with a survival fraction of 0.58 and 0.70 at 75% and 100% of this bacterium’s CFSM (Table 2). These data show that very low cytotoxic effects could be exhibited by CFSM preparations of the probiotic strains except for L. helveticus strain which demonstrated toxicity at high CFSM concentrations (75% and 100%).

Table 2 Cytotoxicity results for the cell free preparations of six probiotic strains on BHK cells
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Additional safety criteria of the probiotic strains

Our preliminary data indicates the ability of two different hosts (mice and poultry) to survive well after oral administration of the whole cultures of the six probiotic strains (data are not shown). Additionally, the analysis of the probiotic genomes against CARD was executed using strict Resistance Gene Identifier (RGI) criteria to decipher the presence of ARGs. Interestingly, genomes of the four Lactobacillus strains were void of ARGS while two ARG families conferring resistance to mupirocin and rifamycin were identified in B. bifidum JCM 1254 and only rifamycin-resistant gene family was found in B. longum DSM 20219 (Supplementary file 1). However, such susceptibility of Bifidobacterium strains to mupirocin and rifamycin requires further experimental validation.

Titration of NDV and IBDV on cell culture

Infectivity titers of NDV and IBDV were determined by infecting CEF cell line with increasing dilutions of virus material, and the highest dilution producing cytopathic effect in 50% of the inoculated cells was determined. The 50% end point dilution expressed as TCID 50/mL was calculated using Reed-Muench formulae. Data showed that CEF infected with NDV and IBDV showed CPE after 4 and 5 days post-infection, respectively. The virus infectivity titer was 106 and 104 TCID 50/mL for NDV and IBDV, respectively.

Effect of probiotics on NDV and IBDV titer

Non-toxic concentration (10%) of CFSM of probiotics was used to treat the CEF cells infected with NDV or IBDV. Reduction in titers of NDV and IBDV in the presence CFSM of probiotics varied with species as shown in Table 3. The percent decrease in virus titer in case of exposure to CFSM of B. bifidum JCM 1254, L. plantarum ATCC 14917, and L. rhamnosus DSM 20021 was 98.3, 98.5, and 97.6%, respectively, followed by B. longum 20219 that caused reduction of 96.3% in NDV titer. The inhibitory effect observed by probiotic strains was much higher in case of NDV as compared to IBDV. Intriguingly, treatment with a mixture of all strains’ CFSM resulted in the highest reduction (99.4 and 83.6% decrease) in titer of NDV and IBDV, respectively. These results suggest the ability of the six probiotic strains to repress in vitro NDV and IBDV infection.

Table 3 Reduction of NDV and IBDV titer by cell free preparations of probiotics
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Mining of host adherence proteins in probiotic genomes

Probiotic Lactobacillus and Bifidobacterium spp. produce host-specific adherence proteins to signal and interact with the host. The whole genomes of the six probiotic strains encoded multiple surface components that adhere to the host. Of these, cell wall anchor domain proteins harboring LPXTG motifs were found in the four Lactobacillus spp., with 5, 2, 1, and 1 motifs for L. helveticus DSM 20075, L. rhamnosus DSM 20021, L. plantarum ATCC 14917, and L. acidophilus ATCC 4356, respectively (Tables 4, 5, 6, and 7). All LPXTG motifs were highly similar to corresponding motifs in other strains of Lactobacillus species. Interestingly, LPXTG motifs showed less identity to similar motifs in marketed probiotic strains. Among these, L. helveticus DSM 20075’s LPXTG motif, EEW68618.1 (product length of 851 amino acids) and L. rhamnosus DSM 20021’s LPXTG motif, and KRK28219.1 (product length of 143 amino acids) are less similar to those in the commercial strains. On the other hand, L. rhamnosus DSM 20021’s LPXTG motif, KRK32331.1, with a high molecular weight had 100% coverage to marketed L. rhamnosus GG but low similarity (67% identity) as shown in Table 5.

Table 4 Host adhesion and interaction proteins in probiotic Lactobacillus helveticus DSM 20075 as determined by genome mining
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Table 5 Host adhesion and interaction proteins in probiotic Lactobacillus rhamnosus DSM 20021 as determined by genome mining
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Table 6 Host adhesion and interaction proteins in probiotic Lactobacillus plantarum ATCC14917 as determined by genome mining
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Table 7 Host adhesion and interaction proteins in probiotic Lactobacillus acidophilus ATCC4356 as determined by genome mining
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Fibronectin binding proteins were found in all probiotic Lactobacillus strains with high similarity to the closest Lactobacillus species. A similar trend was observed with the marketed probiotic Lactobacillus spp. (Tables 4, 5, 6, and 7). A collagen adhesion protein was found only in L. rhamnosus DSM 20021.

Sortases are enzymes which anchor surface proteins in cell wall of Lactobacillus, and Bifidobacterium spp. were encoded in L. helveticus DSM 20075, L. rhamnosus DSM 20021, L. plantarum ATCC 14917, and L. acidophilus ATCC 4356 with 1, 2, 1, and 1 gene products per genome, respectively (Tables 4, 5, 6, and 7).

Bacterial S-layer proteins were encoded in L. helveticus DSM 20075, L. plantarum ATCC 14917, and L. acidophilus ATCC 4356 (1, 1, and 4 gene products per genome, respectively). The S-layer protein, EEW68127.1, in L. helveticus DSM 20075 showed low similarity (54% identity) compared to the closest motif in the marketed probiotic L. helveticus R0052 (Table 4).

The whole genome of L. rhamnosus DSM 20021 encoded 4 WxL domain surface proteins with high similarity to other NCBI’s L. rhamnosus strains and the commercial L. rhamnosus GG. Additionally, ATCC469 strain encoded 1 surface protein, involved in host adherence, called “isopeptide-forming domain-containing fimbrial protein” (Table 5). The protein did not exist in the other three studied Lactobacilli. Glyceraldehyde 3-phosphate dehydrogenase, the enzyme involved in plasma adherence, is encoded in L. plantarum ATCC 14917 genome and exhibited similarity to L. paraplantarum species (Table 6). Twelve mucus-binding proteins were encoded L. acidophilus ATCC4356 with high similarity (> 89% identity) to those encoded by other L. acidophilus strains (Table 7).

Bifidobacterium spp. possesses extracellular structures; pili that play a role in host adherence. Two types of pili, namely tight adherence (Tad) pili and sortase-dependent pili, were found in B. longum DSM 20219 and B. bifidum JCM 1254. Four genes identified in B. longum DSM 20219 genome were found to encode two TadE-like protein, TadA and TadB proteins (Table 8). Interestingly, three sortase A genes were identified in the DSM 20219 genome, in addition to fimbrial isopeptide formation D2 domain-containing protein. Tad proteins and sortase-dependent system were similar in other B. longum strains found in NCBI database (Table 8) but with low similarity to the marketed B. longum JDM301. In a similar fashion, three Tad proteins (TadA, TadE, and TadZ) involved in pili formation were identified in B. bifidum JCM 1254. Additionally, fimbriae formation was elucidated by presence of fimA gene in B. bifidum JCM1254 strain; however, no sortase coding gene was found. Moreover, two collagen binding cell-surface proteins were identified in the JCM 1254 genome (Table 9). All these results document the well-defined capabilities of the probiotic Lactobacillus and Bifidobacterium strains to adhere, colonize, and interact with the human host.

Table 8 Host adhesion and interaction proteins in probiotic Bifidobacterium longum DSM 20219 as determined by genome mining
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Table 9 Host adhesion and interaction proteins in probiotic Bifidobacterium bifidum JCM 1254 as determined by genome mining
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Discussion

Emerging resistance to the current antimicrobial drugs poses a great concern and is considered as a growing public health concern globally. Such resistance has been reported for bacterial, fungal, and viral drugs. Therefore, the search for a novel, safe, and unconventional drugs is of a rising interest. Data obtained from the current work suggest six probiotic strains with potential health benefits that could be exploited in provisional applications in preventive treatments, combating microbial diseases, and formulation of probiotics-enriched diets according to patient phenotypic characteristics. The strains are lactic acid producers and possess genomic determinants involved in bile salt tolerance (Supplementary file 2). These are pivotal criteria for probiotics to survive the gastrointestinal tract. To emphasize the therapeutic use of the six probiotic strains used in this work, the cytotoxicity assessment was conducted. The CFSM of all probiotics demonstrated relatively very low cytotoxicity on BHK-2 cells which encourage their applications for further in vivo studies. Likewise, previous work concluded that the cell viability of Vero cells was around 80% when exposed to probiotic B. longum SPM1205 and SPM1206 and L. ruminis SPM0211 [54]. In addition, cell extract of B. adolecentis SPM0212 had no cytotoxicity on HepG2.2.15 [37]. These findings are in agreement with our results and augment the safety potential of the six strains studied in this work. In addition, genome mining analysis revealed the absence of ARGs in the strains except for Bifidobacterium strains which harbored up to two ARGs. However, this resistance pattern requires validation. For example, putative antibiotic resistance genes were identified in strains of the multispecies VSL#3 marketed probiotic product [45]. However, the strains showed sensitivities to the corresponding antibiotics within the recommended minimum inhibitory concentration values after conducting micro-dilution antibiotic susceptibility testing.

Resistance to drugs pertaining to treat enteric, avian, respiratory, and sexually transmitted viruses is on rise [55]. In the last decade, many studies highlighted the potential of probiotics such as lactic acid bacteria and their bioactive metabolites as antiviral drugs [56, 57]. The protective effect and immune-modulatory activity of probiotics against respiratory viruses in mice was clearly described [56]. The antiviral compounds produced by probiotics were identified as non-proteinaceous such as hydrogen peroxide and lactic acid as well as proteinaceous compounds such as bacteriocins [55]. The antiviral activities of probiotics vary by species, where L. fermentum ACA-DC179, E. faecium PCK38, L. plantarum PCA236, L. pentoses PCA227, B. animalis subsp. Lactis BB-12, L. casei Shirota, and B. longum SP07/3 were examples with the highest antiviral activities [55]. In this work, CFSM of all probiotic strains were investigated for their antiviral activities against NDV and IBDV. The six probiotic strains were able to reduce the viral titer of NDV and IBDV, which might be attributed to the ability of probiotics to produce antiviral metabolites which reduce the pH (lactic acid) or interfere with the adsorption of these viruses to their host cells (bacteriocins) [55] or increasing the CEF resistance against viral infection by reducing viral replication throughout produced exopolysaccharides [58]. However, this assumption needs more investigations. Wang et al. [59] revealed that probiotics could inhibit the replication rate of viruses. The current results suggest beneficial use of probiotics’ cell-free preparations or purified compounds in protecting hosts such as poultry against viral infection. In addition, genome mining revealed presence of numerous cell wall-associated components such as S-layer proteins and affirmed a well-defined adhesive potential to host tissues. These criteria could contribute to the antiviral potential of the strains themselves regardless of their metabolites [60, 61].

Besides the antiviral and safety characteristics observed for the 6 Lactobacillus and Bifidobacterium strains investigated in this work, genome mining analyses revealed the wealth of genomic information that document additional probiotic features of these strains. The whole genome sequences of the strains encoded surface structures that are known to mediate host-microbe interaction and adherence. Long extracellular structures such as pili (Tad and sortase-dependent pili) and fimbriae were encoded in Lactobacillus and Bifidobacterium. Tad pili were found to be essential for gut colonization in B. breve [62]. Sortase-dependent pili that encode for FimB were capable of binding to xylan and other matrices that are important for interaction with the host. Furthermore, sortase-dependent pili proteins expressed in Lactococcus lactis enhanced the adhesion to human enterocytes [63].

LPXTG motifs harbored in cell-surface-associated proteins were encoded in all Lactobacillus strains as well as the marketed probiotic species used for comparison. Cell wall containing LPXTG motifs were found in different Lactobacillus and Bifidobacterium spp. that constitute the VSL#3 marketed probiotics and considered as important players in adherence and interaction with the host [45]. S-layer proteins were found in L. helveticus DSM 20075, L. plantarum ATCC 14917, and L. acidophilus ATCC 4356. These proteins played a role in T cell functioning and signaling dendritic cells [64, 65]. Genes encoding fibronectin binding proteins and collagen adhesins were found in some of the probiotic strains. These proteins were identified to play potential role in host adherence of L. plantarum BP06, L. acidophilus BA05, and B. animalis BL03 [66].

Four WxL domain surface proteins were encoded in L. rhamnosus ATCC7469 and showed similarity to those encoded in the well-studied L. rhamnosus GG. The WxL domains are cell wall-associated proteins and recognized to play a role in cell wall binding in E. faecalis [67]. Glyceraldehyde-3-phosphate dehydrogenase identified in L. plantarum DSM 20021 was also found in L. plantarum LA 318 and proved to mediate high adhesion to human colonic mucin [68]. The whole genome of L. acidophilus ATCC 4356 was rich in mucus-binding proteins. Similarly, putative factors adhering to mucus were identified in L. acidophilus NCFM [69]. Besides the key role of adherence factors in host colonization and interaction, they might modulate the host immune system. Our data showed presence of numerous cell-surface components such as sortase-dependent proteins. A study conducted by Call et al. [70] demonstrated that L. acidophilus sortase gene (srtA) mutant induced lower quantities of IL-12 and TNF-ɑ from the murine DC cells compared to the parent strain. Furthermore, bacterial cell wall elasticity in Lactobacillus and Bifidobacterium strains proved to induce immuno-modulatory cytokines (e.g., IL-12 and INF-Y) as well as nitric oxide production in macrophages but the induction increased with mounting cell wall elasticity [71]. Overall, scientific-driven evidences supported the provisional role of probiotics in influencing innate immune responses, reducing immunity-related metabolic conditions such as obesity [29].

In conclusion, six probiotics belonging to the genera, Lactobacillus and Bifidobacterium, exhibit probiotic traits as summarized in Fig. 1. The strains were found to have very low cytotoxicity and a strong in vitro antiviral activity against two avian viruses; NDV and IBDV which encourage further in vivo studies to expand their applicability. Furthermore, the whole genomes of the probiotic strains were found to be rich in adherence and interaction factors which indicate their well-colonization and host interaction capabilities. These findings indicate the tested probiotic strains to be good candidates in therapeutic purposes and their beneficial use in further in vivo investigations.

Fig. 1
figure 1

Summary of the potential probiotic mechanisms attributed to the studied Lactobacillus and Bifidobacterium strains

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Probiotics and personalized, preventive, and predictive medicine

The interest in personalized medicine is on the rise because there is a need for the patient phenotypic characteristics to determine the right medication at the right dose in an attempt to maximize the treatment efficacy [72]. Probiotics could be exploited in personalized medicine through the development of probiotic supplements according to the patients’ metabolic patterns which vary by lifestyle, diet, environment, and individual genes in each patient. Being a part of the healthy gut microbiome, probiotics could be used for the development of personalized treatment against metabolic syndrome disease(s) which is associated with altered microbiome-host interaction. Additionally, microbiome analysis of the gut microbiota in individuals could be used for predictive diagnosis of metabolic, infectious, or inflammatory diseases provided that high-throughput imaging techniques are implemented. Probiotics could act as the biomarkers for human diseases associated with dysbiosis of the gut microbiota composition and dynamics. However, this will depend on the cost of microbiome sequencing and the availability of the technology throughout the clinical labs. Moreover, the exploitation of probiotics in treatment as alternative to antimicrobial drugs against multidrug resistance bacterial pathogens also showed promising findings [73]. Furthermore, the role of probiotics in preventive medicine has a growing interest and there are accumulative studies that emphasize their role in improving human health [24]. For example, evidences for their ability in prevention and treatment of urinary tract infections [74], and candida vaginitis [75], are promising. Probiotics could be useful in prevention of dental caries, respiratory tract infections, inflammatory bowel disease, and necrotizing enterocolitis [25]. Additional health benefits were reported to improve growth in healthy and malnourished persons. However, the health-promoting effects are dependent on strain type, dosing regimen, and patient’s individual responses.

Findings from the current study support the feasibility of using such probiotic strains in the treatment of microbial infections. Data provided in this work, in addition to our previous study [34], indicate that oral administration of these strains in mice or poultry might protect the corresponding host against bacterial or viral pathogens throughout the antagonistic properties and the adherence capabilities to host tissues. The latter provides colonization resistance and competitive exclusiveness against invading pathogens. Based on currently available literature, taxonomically similar strains to those used in this study were effective in treatment of numerous pathological conditions in clinical trials [32]. Similar benefits could be ascribed to our strains and these encourage further investigations to validate such aspects.

Limitations

Although evidence for the health-promoting functions of probiotics used in this study accumulates, further investigations to emphasize their safety, viability, and efficacy in animal and clinical settings are needed. In addition, optimizing preparation methods (e.g., traditional vs. encapsulation) of probiotics is worth the consideration. Strain-specific properties should be considered when selecting strains for individualized treatments. Despite the growing evidence about effective use of probiotics in treatment of human diseases, legislative rules complicate their way toward approval and commercialization. However, the advancement of current high-throughput technologies could facilitate probiotics’ full characterization and shorten the time needed to gather information about their implications in human health.

Recommendations

  • Evidence-based knowledge of the role of probiotics in preventive and personalized medicine should be considered with caution because the beneficial effect is strain-dependent.

  • Whole genome sequencing of probiotic strains provide privileges to identify, throughout genome mining tools, the safety-related genes, probiotics features of the strains, and their ability to interact with the host. This enables reasonable expectations about the potential probiotic strains considered for animal studies or clinical trials. It also allows the comparison with commercially available strains whose genomes are fully characterized.

  • Screening for probiotic properties under in vitro conditions narrow down the strains that could be exploited for further in vivo experiments.

  • Probiotics that showed promising results under in vitro and in vivo experiments will be good candidates for clinical trials.

  • Evidence-based knowledge about the health-promoting probiotic strains could be gained first through laboratory validation followed by in vivo experiments using animal models.

  • Data regarding the significant role of probiotics in preventive medicine and treatment of several human diseases are accumulating; however, the contribution to personalized medicine needs addition investigations.

  • Metabolic profiling of probiotics individually and within a community of intestinal microbiota is crucial for development and interpretation of biomarkers required for disease diagnostics.

  • Translating the obtained data in the current work into provisional application of the probiotic strains in preventing diseases and design of personalized diets for promoting health is an interesting area of further research.