Precision engineering of the gut microbiome holds promise as an effective therapeutic approach for diseases associated with a disruption in this microbial community. Engrafting a live biotherapeutic product (LBP) in a predictable, controllable manner is key to the consistent success of this approach and has remained a challenge for most LBPs under development. We recently demonstrated high-level engraftment of Bifidobacterium longum subsp. infantis (B. infantis) in adults when co-dosed with a specific prebiotic, human milk oligosaccharides (HMO). Here, we present a cellular kinetic-pharmacodynamic approach, analogous to pharmacokinetic-pharmacodynamic-based analyses of small molecule- and biologic-based drugs, to establish how HMO controls expansion, abundance, and metabolic output of B. infantis in a human microbiota-based model in gnotobiotic mice. Our data demonstrate that the HMO dose controls steady-state abundance of B. infantis in the microbiome, and that B. infantis together with HMO impacts gut metabolite levels in a targeted, HMO-dependent manner. We also found that HMO creates a privileged niche for B. infantis expansion across a 5-log range of bacterial inocula. These results demonstrate remarkable control of both B. infantis levels and the microbiome community metabolic outputs using this synbiotic approach, and pave the way for precision engineering of desirable microbes and metabolites to treat a range of diseases.

Manipulation of the gut microbiome using live biotherapeutic products shows promise for clinical applications but remains challenging to achieve. Here, we induced dysbiosis in 56 healthy volunteers using antibiotics to test a synbiotic comprising the infant gut microbe, Bifidobacterium longum subspecies infantis (B. infantis), and human milk oligosaccharides (HMOs). B. infantis engrafted in 76% of subjects in an HMO-dependent manner, reaching a relative abundance of up to 81%. Changes in microbiome composition and gut metabolites reflect altered recovery of engrafted subjects compared with controls. Engraftment associates with increases in lactate-consuming Veillonella, faster acetate recovery, and changes in indolelactate and p-cresol sulfate, metabolites that impact host inflammatory status. Furthermore, Veillonella co-cultured in vitro and in vivo with B. infantis and HMO converts lactate produced by B. infantis to propionate, an important mediator of host physiology. These results suggest that the synbiotic reproducibly and predictably modulates recovery of a dysbiotic microbiome.

Predictable and sustainable engraftment of live biotherapeutic products into the human gut microbiome is being explored as a promising way to modulate the human gut microbiome. We utilize a synbiotic approach pairing the infant gut microbe Bifidobacterium longum subspecies infantis (B. infantis) and human milk oligosaccharides (HMO). B. infantis, which is typically absent in adults, engrafts into healthy adult microbiomes in an HMO-dependent manner at a relative abundance of up to 25% of the bacterial population without antibiotic pretreatment or adverse effects. Corresponding changes in metabolites are detected. Germ-free mice transplanted with dysbiotic human microbiomes also successfully engraft with B. infantis in an HMO dependent manner, and the synbiotic augments butyrate levels both in this in vivo model and in in vitro cocultures of the synbiotic with specific Firmicutes species. Finally, the synbiotic inhibits the growth of enteropathogens in vitro. Our findings point to a potential safe mechanism for ameliorating dysbioses characteristic of numerous human diseases.

Tractable microbial communities are needed to bridge the gap between observations of patterns of microbial diversity and mechanisms that can explain these patterns. We developed cheese rinds as model microbial communities by characterizing in situ patterns of diversity and by developing an in vitro system for community reconstruction. Sequencing of 137 different rind communities across 10 countries revealed 24 widely distributed and culturable genera of bacteria and fungi as dominant community members. Reproducible community types formed independent of geographic location of production. Intensive temporal sampling demonstrated that assembly of these communities is highly reproducible. Patterns of community composition and succession observed in situ can be recapitulated in a simple in vitro system. Widespread positive and negative interactions were identified between bacterial and fungal community members. Cheese rind microbial communities represent an experimentally tractable system for defining mechanisms that influence microbial community assembly and function.

Consuming diets rich in plant versus animal products changes the microbes found in the human gut within days, with important implications for our health and evolution. Diet influences the structure and function of the gut microbiota in the long term, but it is not clear how rapidly the microbiota is affected by short-term dietary change. Peter Turnbaugh and colleagues studied the effect of transition to a diet consisting entirely of either animal products or plant products on the composition and function of the human gut microbiota. They find that the community changes rapidly, within a single day, overwhelming the pre-existing inter-individual differences in microbiota composition to recapitulate expected patterns of composition and metabolic function for carnivorous and herbivorous mammals. The animal-based diet was associated with higher levels of bile-tolerant microorganisms, including the bacterium Bilophila wadsworthia, which has previously been linked to inflammatory bowel disease. The authors also detected intact foodborne fungi, bacteria and viruses in the distal gut. Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut1,2,3,4,5, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals2, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease6. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

What is cheese? Cheese is a fermented milk product that likely dates back to Neolithic times. Historically serving as a means of preserving milk, today, a fine cheese is viewed as a delicacy rather than a means of survival. Millennia of cheesemaking has led to a diversification of cheese styles and production methods, and more recently, to detailed technical knowledge of the science behind the process. Communities of microbes catalyze the transformation of milk into cheese and remain active participants in the development of a cheese throughout the aging process. How is cheese made? Cheesemaking occurs in three main stages. In the first stage, milk is transformed into solid curds and liquid whey through the coagulation of the milk protein casein. The coagulation of casein is usually accomplished through two complementary methods, acidification and proteolysis. Acidification occurs when lactic acid bacteria ferment the disaccharide lactose, to produce lactic acid. Originally, cheesemakers relied upon naturally occurring lactic acid bacteria in the milk, but today, the process is usually standardized by the addition of domesticated bacterial ‘starter’ cultures, including strains of Lactococcus lactis, Streptococcus thermophilus and Lactobacillus sp. The production of acid by these bacteria causes casein to slowly coagulate. This process is often assisted by the addition of the enzyme chymosin, the active ingredient in rennet. Rennet is traditionally made from an extract of the intestinal lining of a milk-fed calf, which produces the protease chymosin to aid in the digestion of milk. Chymosin removes a negatively charged portion of casein, resulting in the rapid aggregation of casein proteins. In the second stage of cheesemaking, cheesemakers separate the curds, containing the casein and milk fat, from the whey. Depending on the type of cheese, the curds can be heated, salted, pressed, and eventually formed into wheels of various shapes and sizes. Cheese can be eaten fresh at this point, or the wheels can be left to age in a damp, cool place. It is during the aging stage of cheesemaking that cheese is truly transformed — from fresh cheese into the myriad flavors, aromas, and textures of mature cheese. As a normal part of the aging process, starter cultures and non-starter lactic acid bacteria continue to grow and metabolize the interior of the cheese, while the surface of a cheese is colonized by bacteria and fungi that form a multispecies biofilm, termed the ‘rind’ of the cheese (Figure 1). How do microbes impact the flavor, smell, and texture of cheese? Much of the diversity in the flavor, smell, and texture of cheese can be attributed to microbiology. Microbes have a rich assembly of metabolic capacities, and through the production of digestive enzymes and small molecules, microbes contribute to the distinct character of a cheese. However, variations in cheese production can lead to the preferential growth of different groups of microbes. First, the source and treatment (i.e., raw vs. pasteurized) of milk used for cheesemaking can lead to differences in microbial diversity. Subsequently, changes in the pH, salt, moisture, and temperature of a cheese during the initial stages of cheesemaking, or during aging, can dramatically impact the physiology of cheese-associated microbes. The contribution of certain microbes to cheese has been well characterized, and pure cultures of these microbes are commonly used by cheesemakers. Besides the lactic acid bacterial starter cultures, various species of bacteria and fungi can be added to give a cheese very specific characteristics. What is the white fuzzy rind on Camembert? Spores of the filamentous fungus Penicillium camemberti are inoculated into milk during the production of bloomy rind cheeses such as Brie and Camembert. P. candidum is an aerobe and grows preferentially on the surface of the cheese, where it forms a rind made of a dense mat of hyphae (Figure 1A). During growth, proteases are secreted from the hyphae into the cheese. The proteolysis of the casein destroys the structure of the underlying curd, slowly liquefying the cheese and giving Camembert its oozy texture. What is the blue in blue cheese? A related fungus, Penicillium roqueforti, is the key microbe in blue cheese (Figure 1B). While this fungus can be found growing naturally in the limestone caves of southwestern France where the classic cheese, Roquefort, is produced, it can also be inoculated into milk destined to become blue cheese. Because P. roqueforti is a microaerophile, it prefers to grow in crevices created by puncturing a cheese with metal spikes after the wheels are formed. P. roqueforti produces lipases that convert the fats in cheese to peppery free fatty acids and the methyl ketone 2-heptanone, which gives the characteristic blue aroma. The blue pigment seen in blue cheese is produced by P. roqueforti during sporulation. Why are some cheeses so stinky? Washed rind cheeses, such as Epoisses and Limburger, are regularly washed with a brine solution during the aging process. This creates a moist, salty environment on the surface of the cheese in which certain species of fungi and bacteria thrive. One of the best-known species is the actinomycete bacterium Brevibacterium linens (Figure 1C). B. linens contributes to the reddish-orange color typical of these cheeses through the production of carotenoid pigments. Additionally, B. linens metabolizes the casein proteins to a variety of volatile compounds, including amines and sulfur compounds, giving these cheeses their funky, sweaty aromas. The related bacterium B. epidermidis can be found growing on human skin and, not surprisingly, is thought to contribute to body odor. What causes the holes in Swiss cheese? The holes in Swiss cheese result from the growth of the bacterium Propionibacterium freundenreichii (Figure 1D). P. freundenreichii ferments the lactic acid present after the growth of the lactic acid bacteria. The products of this fermentation include propionic acid, which is one of the characteristic flavors of Swiss cheese, and CO2. Because P. freundenreichii prefers anaerobic conditions, growth occurs inside the wheel of cheese, and the CO2 produced during fermentation is trapped and forms the typical bubbles, or holes, found in Swiss cheese. What other microbes are found in cheese? The microbial components of cheese are only partially dictated by the pure cultures inoculated by cheesemakers. A wheel of cheese is home to a whole community of microbes, and the rind in particular may be colonized and dominated by species derived from the local environment rather than by starter cultures. Although the identity and function of these additional species is only beginning to be uncovered, cheesemakers have learned, through trial and error, how to reproducibly cultivate specific communities by manipulating the conditions to which a cheese is subjected during the aging process. Each variety of cheese represents the output of a slightly different microbial community. Because the metabolism of cheese-associated microbes greatly impacts the sensory attributes of cheese, differences in microbial diversity result in tangible changes in flavor, odor, texture, and color that are tracked (and often influenced) by the cheesemaker. Conditions that produce distinct microbial communities have been documented and passed down by generations of cheesemakers. What can we learn from the microbial communities on cheese? Very little is known about how microbes behave in the context of a community. Since studies have recently demonstrated that microbial communities living in and on the human body greatly impact our health, the importance of understanding how microbial communities function and how we can manipulate them is now widely recognized. By studying microbial communities that we already know how to manipulate, like those on cheese, we may readily find answers to questions about which forces are most important in determining succession of species within a microbial community, how species cooperate or compete within a community, and how whole communities respond to perturbations, like invasion by pathogenic species. The adaptation of cheese communities into a laboratory model system can help to delineate the principles that govern microbial communities. Why will cheese be such a good model system for studying microbial communities? A good model system must be simple, easily cultivated in the lab, and reproducible. The rind communities that form a biofilm on the surface of an aged cheese exhibit all of these properties. Because cheese communities have relatively few members, linking species to function will be experimentally practical. Since these communities grow on cheese, a defined substrate, their natural habitat is easily reproduced, and member species may be isolated in the lab. This will make it possible to reconstruct whole communities and observe their development. The simplicity, culturability, and reproducibility of cheese microbial communities will provide a unique, experimentally practical system that will help us understand how microbes live together.