Comparative Genomic Analysis of Probiotic Properties of Host-Adapted Lactobacillus spp. Isolated from Pigs

Abstract

Although probiotics are regarded as AGP alternatives, unproven probiotic strains are frequently used in the animal and functional food industry. Thus it is necessary to understand exact functions and mechanisms of probiotic microbes, and not only health benefits to hosts but also their host adaptation properties should be considered for probiotic selection. Host-specialized microbes show host adaptation properties that were acquired during evolutionary process. Therefore, understanding direction and features of the microbial evolution is important to screen probiotics. Comparative genomic approach helps us to understand functional properties of probiotic microbes and ecology of the microorganisms. In this research, L. salivarius and L. reuteri among probiotic microbes were selected to investigate probiotic properties mentioned above. These two species are important members in the gut microbiota of vertebrates and known as promising probiotic species. Fecal samples were collected from several pig farms in Republic of Korea, and 634 LAB were isolated from the feces. Among them, 284 L. salivarius strains and 104 L. reuteri strains were identified using species-specific multiplex PCR. In study 1, 21 strains were randomly selected among the 284 L. salivarius strains and the draft genomes were generated de novo. These strains were composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade. The pan-genome of L. salivarius strains isolated from humans, pigs and chickens were investigated using all available genomes obtained from NCBI genome database, focusing on host adaptation. MLSA-based phylogenetic clustering showed a distinct categorization of L. salivarius strains depending on their hosts. Among 4,431 orthologs in the pan-genome, 15 host-specific genes and 16 dual-host-shared genes were identified, and several genes associated with host-specific extracellular proteins were identified. 56 extracellular protein encoding genes and 124 orthologs related to EPS production in the pan-genome were also examined, and the distribution and/or nucleotide sequence of these genes are distinguished by the bacterial hosts. Furthermore, some host-specific genes are responsible for energy production and resistance against host stress. In summary, L. salivarius has evolved to adapt to host habitats in three ways, by gaining the abilities for niche adhesion, efficient utilization of nutrients, and resistance against host stress. Furthermore, L. salivarius acquired some genes that have mutualistic properties, contributing to increase the fitness of both the hosts and the microbes during the evolutionary process. It was hypothesized that the microbial genomes have also been affected by the legal prohibition of AGPs, and L. salivarius was investigated in terms of influence of the AGP ban. Several genomic differences between SBPs and SAPs were found, although the number and function of AR genes were not different. SBPs showed larger genome size and a higher number of orthologs than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production and nucleotide synthesis to protect them against antibiotic action. Because these sugars are also used in EPS synthesis, differences in biofilm formation-associated genes were identified. The genes for the production of EPS and cell wall proteins were different in terms of nucleotide sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct resistance strategy against antibiotics. Meanwhile, it may be that the bacteria which have ability to form dense biofilm have existed before the prohibition of AGPs with a small portion, and they have become majority in the L. salivarius population after the AGP ban. Study 2 mainly focused on genetic features of L. reuteri associated with host specificity and their antipathogenic effect. 104 L. reuteri strains were isolated from porcine feces, and 16 strains, composed of eight strains exhibiting the higher antipathogenic effect (HSs) and eight strains exhibiting the lower effect (LSs), were selected for genomic comparison. Draft genomes of the 16 isolates were generated and their pan-genome was investigated together with the 26 NCBI-registered genomes. L. reuteri pan-genome possesses 6,250 orthologs including 890 core genes and 2,081 unique genes. L. reuteri genomes organized six clades with MLSA, and the clade IV includes the 16 isolates. First, we identified six L. reuteri clade IV-specific genes including three hypothetical protein coding genes. The three annotated genes encode transposases and cell surface proteins, indicating that these genes are the result of adaptation to the host epithelia of GIT and suggesting the possibility that these host-specific traits would be acquired by HGT. Differences between group HS and LS were also identified in the pdu-cbi-cob-hem gene cluster, which is essential for reuterin and cobalamin synthesis, and six genes specific to group HS are revealed. While the strains of group HS possessed all genes of this cluster, LS strains have lost many genes of the cluster. cbiD, cbiO, cbiP, sirA, hemB and hemD were present in HSs but absent in in LSs. Based on this result, a genetic marker using four genes were designed and a method using multiplex PCR were developed for selection of antipathogenic L. reuteri strains. The pan-genomic analyses of L. salivarius and L. reuteri revealed several host adaptation traits, including mutualistic properties. This finding is important to understand the evolutionary changes in reciprocal interaction in host-microbe. Furthermore, the host-specific traits of the bacteria may provide a basis for future studies of not only L. salivarius and L. reuteri, but also other symbionts. Comparison in the probiotic traits between the two groups of L. salivarius and L. reuteri showed another aspect of the mutualistic symbionts. L. salivarius strains isolated after the AGP prohibition increased biofilm formation. This result revealed that the ban of AGPs affected to the genetic materials of the bacteria, and contribute to select probiotic strains. The comparison of higher and lower antipathogenic ability of L. reuteri strains showed the genetic differences in reuterin and cobalamin production in the two groups. The studies deepen our knowledge of probiotic bacteria and their beneficial traits and ecology of the probiotic species. Moreover, these studies provide detailed insight for the development of effective probiotics for livestock.