BackgroundThe Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ).ResultsCommensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ.ConclusionCombining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0735-2) contains supplementary material, which is available to authorized users.

Streptococcus gallolyticus subsp. gallolyticus, formerly classified as S. bovis biotype I, is an increasing cause of bacteremia and infective endocarditis in the elderly. The physiopathology of infective endocarditis is poorly understood and involves immune and coagulation systems. In this study, we found that S. gallolyticus subsp. gallolyticus activates the human contact system, which in turn has two consequences: cleavage of high-molecular-weight kininogen (HK) resulting in release of the potent pro-inflammatory peptide bradykinin, and initiation of the intrinsic pathway of coagulation. S. gallolyticus subsp. gallolyticus was found to bind and activate factors of the human contact system at its surface, leading to a significant prolongation of the intrinsic coagulation time and to the release of bradykinin. High-affinity binding of factor XII to the bacterial Pil1 collagen binding protein was demonstrated with a KD of 13 nM. Of note, Pil1 expression was exclusively found in S. gallolyticus subsp. gallolyticus, further supporting an essential contribution of this pilus in virulence.

Research and marketing of probiotics demand holistic strain improvement considering both the biotic and abiotic gut environment. Here, we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donor’s gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole-genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport, and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205, and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization under colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence. IMPORTANCE Improvement of bacterial strains in regard to specific abiotic environmental factors is broadly used to enhance strain characteristics for processing and product quality. However, there is currently no multidimensional probiotic strain improvement approach for both abiotic and biotic factors of a colon microbiota. The continuous PolyFermS fermentation model allows stable and reproducible continuous cultivation of colonic microbiota and provides conditions akin to the host gut with high control and easy sampling. This study investigated the suitability of PolyFermS for adaptive evolutionary engineering of a probiotic model organism for lactobacilli, Lactiplantibacillus plantarum, to an adult human colonic microbiota. The application of PolyFermS controlled gut microbiota environment led to adaptive evolution of L. plantarum strains for enhanced gut colonization characteristics. This novel tool for strain improvement can be used to reveal relevant factors involved in gut microbiota colonization and develop adapted probiotic strains with improved functionality in the gut.

The human gut microbiota (HGM) plays a pivotal role in health and disease. Consequently, nutritional and medical research focusing on HGM modulation strategies as a means of improving host health is steadily increasing. In vitro HGM fermentation models offer a valid complement to human and animal studies when it comes to the mechanistic exploration of novel modulation approaches and their direct effects on HGM composition and activity, while excluding interfering host effects. However, in vitro cultivation of HGM can be challenging due to its high oxygen sensitivity and the difficulties of accurately modeling the physio-chemical complexity of the gut environment. Despite the increased use of in vitro HGM models, there is no consensus about appropriate model selection and operation, sometimes leading to major deficiencies in study design and result interpretation. In this review paper, we aim to analyze crucial aspects of the application, setup and operation, data validation and result interpretation of in vitro HGM models. When carefully designed and implemented, in vitro HGM modeling is a powerful strategy for isolating and investigating biotic and abiotic factors in the HGM, as well as evaluating their effects in a controlled environment akin to the gut. Furthermore, complementary approaches combining different in vitro and in vivo models can strengthen the design and interpretation of human studies.

Research and marketing of probiotics demand for holistic strain improvement considering both, the biotic and abiotic gut environment. Here we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donors gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205 and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization in colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence.

Streptococcus infantarius subsp. infantarius (Sii) has been identified as predominant lactic acid bacteria in spontaneously fermented dairy products (FDPs) in sub-Saharan Africa including Côte d'Ivoire. However, Sii belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC). Most SBSEC members are assumed to be involved as opportunistic pathogens in serious diseases in both humans and animals. A population-based cross-sectional survey, including 385 participants was conducted in Korhogo, northern Côte d'Ivoire, to identify risk factors for Sii fecal carriage, including consumption of local FDPs. A structured questionnaire was used to gather participant's socio-demographic and economic characteristics, their relation to livestock and dietary habits. In addition, fresh stool and milk samples were collected. The identification of Sii was done using a SBSEC-specific PCR assay targeting 16S rRNA and groEL genes. The overall prevalence of SBSEC and Sii carriage was 23.2% (confidence interval CI 95% = 18.9–27.5) and 12.0% (CI 95% = 8.4–15.5) for stool, respectively. Prevalence of Sii was significantly higher in consumers of artisanal butter compared with non-consumers (57.1% vs 10.1%, odds ratio OR: 11.9, 95% CI: 3.9–36.6), as well as in persons handling livestock (OR = 3.9; 95% CI = 1.6–9.3) and livestock primary products (OR = 5.7; 95% CI = 2.3–14.3). The closer contact with livestock was a risk factor for Sii fecal carriage. Sii strains were isolated from fresh and fermented milk products with a prevalence of 30.4% and 45.4%, respectively. Analysis of Sii population structure through the SBSEC multi locus sequence typing assay revealed a close relationship across human and dairy isolates, possibly linked to a Kenyan human isolate. All these outcomes underline the interest of in-depth investigations on the ecology, potential reservoirs and pathways of contamination by Sii at the human-animal-environment interface in comparison to yet to be collected data from Europe, Asia and the Americas to further elucidate the various roles of Sii.

Background Auto-aggregation is a desired property for probiotic strains because it is suggested to promote colonization of the human intestine, to prevent pathogen infections and to modulate the colonic mucosa. We recently reported the generation of adapted mutants of Lactiplantibacillus plantarum NZ3400, a derivative of the model strain WCFS1, for colonization under adult colonic conditions of PolyFermS continuous intestinal fermentation models. Here we describe and characterize the emerge of an auto-aggregating phenotype in L. plantarum NZ3400 derivatives recovered from the modelled gut microbiota. Results L. plantarum isolates were recovered from reactor effluent of four different adult microbiota and from spontaneously formed reactor biofilms. Auto-aggregation was observed in L. plantarum recovered from all microbiota and at higher percentage when recovered from biofilm than from effluent. Further, auto-aggregation percentage increased over time of cultivation in the microbiota. Starvation of the gut microbiota by interrupting the inflow of nutritive medium enhanced auto-aggregation, suggesting a link to nutrient availability. Auto-aggregation was lost under standard cultivation conditions for lactobacilli in MRS medium. However, it was reestablished during growth on sucrose and maltose and in a medium that simulates the abiotic gut environment. Remarkably, none of these conditions resulted in an auto-aggregation phenotype in the wild type strain NZ3400 nor other non-aggregating L. plantarum, indicating that auto-aggregation depends on the strain history. Whole genome sequencing analysis did not reveal any mutation responsible for the auto-aggregation phenotype. Transcriptome analysis showed highly significant upregulation of LP_RS05225 (msa) at 4.1–4.4 log2-fold-change and LP_RS05230 (marR) at 4.5–5.4 log2-fold-change in all auto-aggregating strains compared to non-aggregating. These co-expressed genes encode a mannose-specific adhesin protein and transcriptional regulator, respectively. Mapping of the RNA-sequence reads to the promoter region of the msa-marR operon reveled a DNA inversion in this region that is predominant in auto-aggregating but not in non-aggregating strains. This strongly suggests a role of this inversion in the auto-aggregation phenotype. Conclusions L. plantarum NZ3400 adapts to the in vitro colonic environment by developing an auto-aggregation phenotype. Similar aggregation phenotypes may promote gut colonization and efficacy of other probiotics and should be further investigated by using validated continuous models of gut fermentation such as PolyFermS.

Humans ingest many microorganisms, which may colonize and interact with the resident gut microbiota. However, extensive knowledge about host-independent microbe-microbe interactions is lacking. Here, we investigated such colonization process using a derivative of the model probiotic Lactiplantibacillus plantarum WCFS1 into continuously cultivated gut microbiota in the intestinal PolyFermS fermentation model inoculated with five independently immobilized human adult fecal microbiota. L. plantarum successfully colonized and organized itself spatially in the planktonic, that is, the reactor effluent, and sessile, that is, reactor biofilm, fractions of distinct human adult microbiota. The microbiota carrying capacity for L. plantarum was independent of L. plantarum introduction dose and second supplementation. Adult microbiota (n = 3) dominated by Prevotella and Ruminoccocus exhibited a higher carrying capacity than microbiota (n = 2) dominated by Bacteroides with 105 and 103 CFU/ml of L. plantarum, respectively. Cultivation of human adult microbiota over 3 months resulted in decreased carrying capacity and correlated positively with richness and evenness, suggesting enhanced resistance toward colonizers. Our analyses ultimately allowed us to identify the fermentation metabolite valerate as a modulator to increase the carrying capacity in a microbiota-independent manner. In conclusion, by uncoupling microbe-microbe interactions from host factors, we showed that L. plantarum colonizes the in vitro colonic community in a microbiota-dependent manner. We were further able to demonstrate that L. plantarum colonization levels were not susceptible to the introduction parameters dose and repeated administration but to microbiota features. Such knowledge is relevant in gaining a deeper ecological understanding of colonizer-microbiota interactions and developing robust probiotic strategies.