The 198 isolates of the isolated Lactobacillus belong to four species: L. fermentum, L. brevis, L. oris, and L. vaginalis. It has been reported that L. fermentum and L. rhamnosus were the most frequent species from breast milk at the level of species (23). In the present study, L. fermentum was the dominant species, which was the same as the results obtained by Soto et al. (24). Moreover, the present study also found that L. fermentum had 100% of relative abundance in colostrum. However, Ozgun et al. revealed that L. brevis was frequently found from colostrum samples from Turkey (25). This may be related to differences in region, the lifestyle and dietary habits of the mother. For colostrum A to mature milk B, the number and species of Lactobacillus isolated showed an uptrend, mature milk B reached the highest level, and showed a downside from mature milk B to late milk E. Similar results were reported by Solis et al., who also found that the Lactobacillus spp. in mature milk (10–90 postpartum days) has higher isolation ratio and more species than colostrum (1 day) (26). It has been reported that the breast milk composition influence microbiota (27). Therefore, we can speculate that the growth of some Lactobacillus may be related to changes of nutrient composition in breast milk during different lactation periods. And mature milk is more suitable for Lactobacillus multiplication. In the present study, four isolates of L. vaginalis were rarely isolated from breast milk of stages B and C, which is seldom reported in other studies. Ana et al. detected L. vaginalis in only 2 of the 27 breast milk samples (24). This may due to its harsh survival requirement or the introduction of new technologies, such as metagenomics and sequencing, which allowed the rapid development of Lactobacillus isolation. Lactobacillus are notoriously widespread in the skin, mouth cavity, gastrointestinal and vaginal cavities of humans (28). As previously reported, L. vaginalis was usually isolated from the vaginas of women (29). As demonstrated by Ramsay et al., a degree of reflux into the mammary duct during breast-feeding may facilitate the exchange of bacteria between mammary glands and the oral cavity of infants who acquire bacteria from the vaginal microbiome at birth (27). This shows that the presence of L. vaginalis in breast milk may be associated with vertical transmission during vaginal birth and breastfeeding.
Since the probiotic characteristics of Lactobacillus are specific to strains, the feasibility of exploring new strains is determined by these probiotic characteristics (30). The authors found that the isolates had strong antimicrobial activity. Notably, the KM66 exhibited a broad antimicrobial spectrum against all indicator pathogens and exhibited the broadest antimicrobial spectrum against E. coli (EPEC), E. coli (ETEC), L. monocytogenes, and S. Typhimurium. Mojgani et al. reported that L. brevis LB32 isolated from ewes’ milk in Iran also exhibited a wide spectrum of inhibition against L. monocytogenes and S. Typhimurium (31). However, the KM15 with the same genotype as KM66 failed to inhibit S. Enterica. The Y3 (L. fermentum) exerted inhibition against all indicator pathogens. This result differs with the report done by Olivares et al., which indicated that L. fermentum CECT5716 isolated from human milk exhibited the suppression of L. monocytogenes and E. coli (ETEC), but failed to inhibit the growth of E. coli (EHEC) (32). The mechanism of the different antibacterial activities of these strains is largely determined by their different secreted antibacterial substances and the competition with pathogenic bacteria on nutrition and adhesion of intestinal epithelial cells (33–35).
The risk of Lactobacillus transmitting antibiotic-resistant genes to intestinal pathogens cannot be ignored. With regard to antibiotic resistance, 95% of isolates exhibited antibiotic resistance to vancomycin and co-trimoxazole, though the majority of isolates were found susceptible to other antibiotics. These results are similar with other research (36,37). Mohammadi et al also showed that 94% of the Lactobacillus isolated from human milk were resistant to vancomycin (38). It has been reported that resistance to vancomycin and co-trimoxazole in certain Lactobacillus strains is not a safety issue since it is encoded by chromosomes rather than acquired, and therefore not transmissible (39). For safety assessment, the resistance to vancomycin can be used as a useful criterion for screening safe probiotic strains of Lactobacillus (40).
Ninety percent of candidates were tolerant to the lysozyme, suggesting that Lactobacillus had high lysozyme tolerance, which was confirmed by other authors (16, 41). However, only 3 isolates were substantially resistant to acid pH 3.0 and 0.3% of bile salt, respectively: KM66 (survival: 61.078%, 75.291%), KM147 (61.077%, 62.940%), and Y3 (55.205%, 65.049%). It indicate that these isolates could survive in digestive tract. However, the KM26 with the same genotype as KM66 exhibited the lowest survival rate of 31.33% and 29.74% correspondingly at acid pH 3.0 and 0.3% of bile salt. The strains of L. fermentum isolated from human gastrointestinal tracts had relatively better acid tolerance in comparison to other species of Lactobacillus (22). Shokryazdan et al. also reported that the L. fermentum HM2 from human milk showed high acid tolerance at pH 3.0 with a resistance of 72.44%, but exhibited low tolerance to 0.3% of bile salt with a resistance of 20.42% (35), proving the specificity of the tolerance of strains. As reported, the L. fermentum CECT5716 isolated from breast milk was added as the probiotic and was found to reduce the incidence of gastrointestinal infection in infants after six months of continuous feeding (42). Additionally, Albesharat et al. demonstrated that L. fermentum, L. brevis, and L. oris were found in the mothers’ milk and the faeces of the corresponding babies (43). In this study, the candidates are expected to colonize the intestine and inhibit the infection of pathogenic bacteria in infants.