Generally, lactic acid bacteria (LAB) are the main group of probiotics used for humans and animals. In the food fermentation, they play a significant role by inhibiting the growth of spoilage/pathogenic microorganisms, and by producing fermented food products with desired flavor, aroma, and texture.
Usually, the probiotic microorganisms are screened from food and nonfood sources. The nonfood sources include gastrointestinal tract, as the main nonfood source, honeycomb, soil and plant surface. On the other hand, the food sources are represented by fermented dairy, meat and vegetable products, and fruit juices.
In the present study, after performing Gram staining, catalase test and blood hemolysis test, the DNA fingerprinting and 16S rRNA gene sequencing were applied to identify 16 acid and bile salt-tolerant bacteria isolated from different food sources. The results revealed that, 43.75% were identified as Enterococcus mediterraneensis isolated from rayeb milk, kareish cheese, and frankfurte. Also, 31.25% were classified as Lactobacillus fermentum isolated from rayeb milk, yoghurt, and frankfurter. Both Streptococcus lutetiensis and Bacillus circulans represented 12.5% of the total isolates. Enterococcus mediterraneensis was first isolated and identified in 2019 from the stool of a 39-year-old male Pygmy in the Democratic Republic of Congo [21]. There are no previous studies reporting isolation of E. mediterraneensis from Egyptian sources. Therefore, this study is considered as the first one to isolate Enterococcus mediterraneensis in Egypt and to characterize its probiotic properties worldwide.
Streptococcus lutetiensis is belonging to Streptococcus bovis/Streptococcus equinus complex (SBSEC) which is a non-enterococcal group D Streptococcus spp. complex. The strains of SBSEC are commensal colonizers of the gastrointestinal tract of humans and animals including ruminants as cattle, sheep, goats, and camels. Some strains of SBSEC have been associated with different diseases as endocarditis, bacteremia, biliary tract, prosthetic joint infections, meningitis, and diarrhea. Additionally, some strains are considered as important species having a main role in the quality of fermented food products. Moreover, some SBSEC strains as Strep. lutetiensis and Strep. gallolyticus subsp. macedonicus are consumed as a part of the daily diet. Consequently, they are considered to be safe for human consumption [22, 23]. For blood hemolysis activity, some strains are gamma-hemolytic (non-hemolytic), which agreed with the results of the present study, and some exhibit alpha-hemolytic activity.
The representatives of various Bacillus species have a long history of safe use as probiotics. Globally, there is a variety of commercial formulations containing Bacillus spp. to be used as probiotics [24]. Bacillus circulans, reported to cause human infection, is a member of the Bacillus subtilis group [25]. Most species of this group exhibit β-hemolytic activity. In this study, Bacillus circulans was isolated from frankfurter and exhibited γ-hemolysis on blood agar. This result was in agreement with the findings of Alebouyeh et al. [26] who isolated nonhemolytic B. circulans from a 62-year-old patient with 4 years of unknown end-stage renal disease. It is known that Bacillus circulans is an opportunistic pathogen found in soil, sewage, and food. Also, many previous studies isolated B. circulans from cases of meningitis, prosthetic heart valve [27], endocarditis, endophthalmitis [28], and wound infection. There are other reports indicating that B. circulans is a causative agent of sepsis in immunocompromised hospitalized patients [24].
This study reported the isolation of Clavispora lusitaniae from kareish cheese. Clavispora lusitaniae, which is also known as Candida lusitaniae, could be isolated from different sources as digestive tract, fruit juices, citrus peel, and milk from cow infected with mastitis. Generally, Clavispora lusitaniae is considered as a nosocomial pathogen [29].
Generally, the microorganisms are considered as safe and beneficial probiotics for human and animal use after their proper identification and characterization. Consequently, in vitro characterization of safety and functional properties is extremely imperative for the selection of highly effective probiotic strains. In the current study, the functional assays to evaluate the probiotic efficiency of isolated B. circulans and Clavispora lusitaniae were not performed because they are stated as microbial pathogens as was previously mentioned.
Lacking the hemolytic activity is one of the most important safety characteristics recommended by FAO/WHO (2002) for probiotic microorganisms to be considered as food grade bacteria [30]. Actually, all lactic acid bacterial isolates (14 isolates) were nonhemolytic isolates. The absence of hemolytic activity of isolated E. mediterraneensis was in agreement with the previous study of Takakura et al. [21].
Evaluating the antibiotic susceptibility is considered as the second important safety aspect regarding employing the bacteria as probiotics in food and animal feed. The results revealed that the isolates of E. mediterraneensis were susceptible to ampicillin and ceftolozane inhibiting the cell wall biosynthesis, and to neomycin inhibiting the protein synthesis, unlike the other enterococci reported by Miller et al. [31] to have innate resistance to antibiotics of β-lactams and aminoglycosides. All Lb. fermentum isolates exhibited susceptibility to ceftolozane, neomycin, and sulphamethoxazole inhibiting the cell wall and protein biosynthesis and folic acid metabolism, respectively. These results were in agreement with previous studies of Danielsen and Wind [32] and Abriouel et al. [33]. The two isolates of Strep. lutetiensis displayed sensitivity to antibiotics with mode of action to inhibit the biosynthesis of cell wall and proteins.
Antibiotic resistance of probiotics and absence of transferable antibiotic resistance genes, that could be transferred horizontally to other bacteria, are imperative to avoid the risk of prevalence of antibiotic resistance genes in the environment and to confirm the safety of probiotic application as food and feed additives [34]. European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) defined the multidrug resistance (MDR) as the resistance to at least one agent in three or more antimicrobial categories [35]. According to this definition, E. mediterraneensis isolates (L3, L11, L12, and L16), all isolated Lb. fermentum and Strep. lutetiensis are regarded as multidrug-resistant bacteria. Generally, lactobacilli are known to have intrinsic resistance to vancomycin [36, 37]. The genes encoding its resistance are located on the chromosome which indicates these genes are not transferred horizontally. On the other hand, genes encoding the tetracycline resistance are often located on the conjugative plasmids [38]. Therefore, they could be transferred.
Phenol and its derivatives are known to have antibacterial and antifungal activity. Therefore, evaluating the resistance of potential probiotics to phenol is significant to be applied in animal and fish feeding as these compounds are produced in their intestine by bacterial deamination of aromatic amino acids liberated during digestion of dietary proteins [39]. In the present study, the highest survival rate to 0.4% phenol was recorded with E. mediterraneensis (L16), Lb. fermentum (L9), and Strep. lutetiensis (L14).
Survival in the gastrointestinal juice, cell surface hydrophobicity (CSH), and capability to auto-aggregate are the foremost selective traits of potential probiotics to be functionally effective in the host. Evaluating the performance of probiotic candidates in simulated gastrointestinal environment is essential to sufficiently predict their in vivo behavior as without this property the microorganisms will not be functionally influential [17]. Some studies have evaluated the resistance of probiotics to the gastrointestinal juices through using the gastric and intestinal juices individually [16, 40]. In this study, the successive gastric and intestinal digestion was employed to simulate the physiological conditions of human and animal gastrointestinal digestion. The simulated gastric conditions were characterized by the presence of 0.3% bile salt and 0.1% pepsin enzyme in acidic conditions (pH 2.5), whereas the simulated intestinal conditions were represented by the presence of 1% pancreatin containing trypsin, lipase, protease, and amylase enzymes in higher pH of 6.8. The tolerance of potential probiotics, used in fish aquaculture and animal feeding, to bile salt is substantial not only to confirm their ability to survive in the indigenous bile salt present naturally in fish and animal intestine, but also to that added to animal and fish feeds. Recently, the plant feed ingredients, supplemented with bile salt, are employed to replace fishmeal and fish oil in feed production. This supplementation is very essential because some compounds for bile salt synthesis, as cholesterol and taurine, are usually insufficient in plant feed ingredients [41]. The results revealed that some isolates have high survivability that reached 23.9 ± 1.85 and 32.73 ± 0.84% for Lb. fermentum (L8) and E. mediterraneensis (L2), respectively. Conversely, the low survival rates of 2.01 ± 0.01 and 1.35 ± 0.06% were recorded for Lb. fermentum (L9) and E. mediterraneensis (L16), correspondingly. The low survivability may be attributed to the antimicrobial effect of bile salts which causes permeabilization of the bacterial cell membrane and leakage of cytosol consequently. Some researchers reported that the effect of bile salt on the bacterial cytoplasmic membrane depends on its concentration. The high concentrations dissolve membrane lipids, causing leakage of cell materials and cell death. Low concentrations have less undesirable effects on the membrane fluidity and permeability by changing membrane proteins or increasing transmembrane divalent cation flow [42]. According to Botta et al. [17] who reported that the microorganisms with ODS less than 0.00001%, after sequential transfer from gastric to intestinal juice, are not considered resistant to the gastrointestinal conditions, all E. mediterraneensis, Lb. fermentum, and Strep. lutetiensis isolates have an extremely considerable resistance as the lowest ODS value was 1.35 ± 0.06%.
The high cell surface hydrophobicity and strong auto-aggregation capability are considered as essential requirements of probiotics to ensure strong colonization and adhesion to intestinal epithelium of the host to provide their health benefits. Also, the strong adhesion to mucosal surfaces and epithelial cells of the gastrointestinal tract allows probiotics to overcome the gastric motility and therefore enhances the interactions between probiotic bacteria and host [43]. Generally, the cell surface hydrophobicity is different between bacterial species, but there are numerous compounds playing a main role in the bacterial CSH. These compounds include lipoteichoic acid, core oligosaccharides, outer membrane proteins and lipids, surface fibrils and several fimbriae [43]. Hydrophobicity is likely due to a complex interaction between positively charged, negatively charged, hydrophilic and hydrophobic components on the bacterial surface [44]. The studies of Jena et al. [16] and Abdulla et al. [44] reported that the bacterial strains, with more than 40% hydrophobicity, will be considered hydrophobic. According to their findings, E. mediterraneensis (L3), Lb. fermentum (L5), and Strep. lutetiensis (L13) could be considered as hydrophobic isolates as their CSH, ranging from 37.87 ± 9.71 to 39.79 ± 2.87%, is slightly less than 40%.
Bacterial auto-aggregation is defined as the ability of bacteria of the same strain to bind to themselves. This phenomenon is observed clearly through the formation of bacterial clumps that precipitate at the tube bottom. Generally, the auto-aggregation is mediated by exopolysaccharide and surface proteins as extracellular serine/threonine-rich protein of Lb. plantarum NCIMB 8826 [45] and S-layer proteins of Lb. acidophilus M92 [46]. The current study confirmed the findings of other studies reported that the self-aggregation increases with extending the incubation time [16, 44]. The greatest auto-aggregation ability, higher than 50%, was recorded with 71.43% of isolated LAB. In another study, the strongest auto-aggregation ability reached 47.2 ± 2.4% [16].
Although some studies [44, 47, 48] reported the direct correlation between bacterial cell surface hydrophobicity and auto-aggregation capability, the results of this study do not support the hypothesis as relation of the auto-aggregation and CSH was not characterized.
The auto-aggregation and co-aggregation (aggregation between genetically different strains) are considered as key properties of probiotics to prevent colonization of gastrointestinal tract with pathogens. This is due to the formation of biofilms of auto-aggregating bacteria on the intestinal mucosa and intercellular adhesion between co-aggregating bacteria and microbial pathogens [49]. Thence, aggregation could be counted as one of the defense mechanisms of host for anti-infection. In this work, the E. mediterraneensis isolates (L2, L12, and L15) with high auto-aggregation ability exhibited high co-aggregation capability with Sal. typhimurium, E. coil O157:H7, S. aureus, and B. cereus in a range of 55.69 ± 2.42 to 62.95 ± 1.35, 39.73 ± 2.02 to 53.53 ± 1.33, 41.31 ± 3.3 to 44.29±2.96 and 34.19 ± 2.75 to 49.68 ± 2.65%, respectively. Also, the Lb. fermentum isolates (L8, L9, and L10) with high auto-aggregation ability exhibited high co-aggregation with Sal. typhimurium, E. coil O157:H7, S. aureus, and B. cereus in a range of 42.84 ± 5.24 to 49.45 ± 1.25, 27.94 ± 1.5 to 35.85 ± 0.77, 38.61 ± 2.15 to 45.12 ± 3.56 and 45.97 ± 1.97 to 48.91 ± 1.25%, individually. The isolate Strep. lutetiensis (L14) displayed high auto-aggregation of 61.74 ± 1.8% and co-aggregation of 45.16 ± 0.63, 45.76 ± 1.92, 37.32 ± 1.14, and 40.31 ± 2.84% with Sal. typhimurium, E. coil O157:H7, S. aureus, and B. cereus, correspondingly. Thus, these isolates possessing the strong ability to auto-aggregate and co-aggregate pathogens could be valuable to the intestinal health.
The relation between probiotics and pathogenic bacteria depends on the co-aggregation and antimicrobial activity of probiotics to inhibit pathogens. The isolates of prospective probiotic LAB should exhibit manifest antimicrobial activities against pathogenic bacteria causing diseases in human and animal gut to improve the host health and to balance the gut microbiota. In this study, 85.71 and 50% of E. mediterraneensis and Strep. lutetiensis isolates, respectively displayed antibacterial activity against all selected bacterial pathogens. For Lb. fermentum isolates, 40 and 20% had effect against B. cereus and E. coli O157: H7, correspondingly. Furthermore, 60% were active against both Sal. typhimurium and S. aureus. The antibacterial activity of extracts prepared from LAB was evaluated to specify the antimicrobial agents. No inhibitory effect was observed from extract 3 prepared from E. mediterraneensis (L1 and L2), (L12), and Strep. luteliensis (L14) against all tested pathogens, Sal. typhimurium and B. cereus, respectively. Absence of the antagonistic activity of the third extract indicates the antimicrobial effect attributed mainly to organic acids only or organic acids and H2O2 as organic acids are present in the first and second extracts but H2O2 is found in the first extract only. According to the obtained results, 57.14% of E. mediterraneensis isolates (L11, L12, L15, and L16) could be considered bacteriocinogenic against E. coli O157:H7, B. cereus, and S. aureus, whereas 42.86% (L11, L15, and L16) was bacteriocinogenic against Sal. typhimurium. For Lb. fermentum, 60% (L8, L9, and L10) could be characterized as bacteriocinogenic isolates against Sal. typhimurium and S. aureus, 40% (L8 and L9) against B. cereus and finally 20% (L9) against E. coli O157:H7. Also, Strep. lutetiensis (L14) could be specified as bacteriocinogenic against all tested bacterial pathogens. Many studies demonstrated the antagonistic effect of LAB against different pathogens through production of various antimicrobial agents including carbon dioxide, hydrogen peroxide, organic acids, and bacteriocins [17, 40, 43].