1 Cross-feeding between Bifidobacterium infantis and Anaerostipes caccae on 2 lactose and human milk oligosaccharides 3 4 Loo Wee Chia1, Marko Mank2, Bernadet Blijenberg2, Roger S. Bongers2, Steven 5 Aalvink1, Kees van Limpt2, Harm Wopereis1,2, Sebastian Tims2, Bernd Stahl2, Clara 6 Belzer1*#, Jan Knol1,2* 7 * these authors contributed equally 8 1 Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 9 6708 WE Wageningen, the Netherlands. 10 2 Nutricia Research, Uppsalalaan 12, 3584 CT Utrecht, the Netherlands. 11 12 Running Head: Microbial cross-feeding in infant gut 13 14 # Address correspondence to [email protected]. 15 16 Conflict of Interest statement: 17 This project is financially supported by Nutricia Research. MM, BB, RB, HW, KvL, ST, 18 BS and JK are employed by Nutricia Research. 1 19 Abstract 20 The establishment of the gut microbiota immediately after birth is a dynamic process 21 that may impact lifelong health. At this important developmental stage in early life, 22 human milk oligosaccharides (HMOS) serve as specific substrates to promote the 23 growth of gut microbes, particularly the group of Actinobacteria (bifidobacteria). Later 24 in life, this shifts to the colonisation of Firmicutes and Bacteroidetes, which generally 25 dominate the human gut throughout adulthood. The well-orchestrated transition is 26 important for health, as an aberrant microbial composition and/or SCFA production 27 are associated with colicky symptoms and atopic diseases in infants. Here, we study 28 the trophic interactions between an HMOS-degrader, Bifidobacterium longum subsp. 29 infantis and the butyrogenic Anaerostipes caccae using carbohydrate substrates that 30 are relevant in this early life period, i.e. lactose and HMOS. Mono- and co-cultures of 31 these bacterial species were grown at pH 6.5 in anaerobic bioreactors supplemented 32 with lactose or total human milk carbohydrates (containing both lactose and HMOS). 33 A. cac was not able to grow on these substrates except when grown in co-culture 34 with B. inf, leading concomitant butyrate production. Cross-feeding was observed, in 35 which A. cac utilised the liberated monosaccharides as well as lactate and acetate 36 produced by B. inf. This microbial cross-feeding is indicative of the key ecological role 37 of bifidobacteria in providing substrates for other important species to colonise the 38 infant gut. The symbiotic relationship between these key species contributes to the 39 gradual production of butyrate early in life that could be important for host-microbial 40 cross-talk and gut maturation. 41 2 42 Importance 43 The establishment of a healthy infant gut microbiota is crucial for the immune, 44 metabolic and neurological development of infants. Recent evidence suggests that 45 an aberrant gut microbiota early in life could lead to discomfort and predispose 46 infants to the development of immune related diseases. This paper addresses the 47 ecosystem function of two resident microbes of the infant gut. The significance of this 48 research is the proof of cross-feeding interactions between HMOS-degrading 49 bifidobacteria and a butyrate-producing microorganism. Bifidobacteria in the infant 50 gut that support the growth and butyrogenesis of butyrate-producing bacteria, could 51 orchestrated an important event of maturation for both the gut ecosystem and 52 physiology of infant. 53 54 Keywords 55 Bifidobacteria, butyrate, Lachnospiraceae, microbiome, pH 56 3 57 Introduction 58 The succession of microbial species in the infant gut microbiota is a profound 59 process in early life (1, 2), which coincides with the important development of the 60 immune, metabolic and neurological systems (3-5). At this developmental stage, 61 human milk is recognised as the best nourishment for infants (6). Human milk 62 contains a range of microbial active components and among all human milk 63 oligosaccharides (HMOS) have a vital role in the development of the infant gut 64 microbiota (7). HMOS are complex carbohydrates composed of a lactose core, which 65 may be elongated by N-acetylglucosamine (GlcNAc), galactose and/or decorated 66 with fucose and/or sialic acid residues (8). The composition of HMOS in human milk 67 is highly individual and driven by maternal genetic factors and varies with the phases 68 of lactation (9). 69 The majority of the HMOS escape digestion by the host’s enzymes in the 70 upper gastrointestinal tract (10). HMOS confer important physiological traits by acting 71 both as a decoy for the binding of pathogenic bacteria and viruses, and as a prebiotic 72 to stimulate the growth and activity of specific microbes in the infant gut (11). These 73 complex carbohydrates exert therefore a selective nutrient pressure to promote the 74 HMOS-utilising microbes, especially bifidobacteria belonging to the Actinobacteria 75 phylum (12). Bifidobacteria are specifically adapted to utilise HMOS by employing an 76 extensive range of glycosyl hydrolases and transporters, which leads to their 77 dominance in the infant gut (13). Upon weaning, the relative abundance of 78 bifidobacteria decreases with the increase of Firmicutes and Bacteroidetes phyla, 79 whilst the gut microbial diversity increases (14). 80 The early dominance of bifidobacteria could be important for the maturation of 81 the overall microbial community. In healthy children, the relative abundance of 4 82 bifidobacteria is positively associated with the butyrate-producing Firmicutes from the 83 family of Lachnospiraceae (also known as Clostridium cluster XIVa) and 84 Ruminococcaceae (also known as Clostridium cluster IV) (15). This butyrogenic 85 community often presents at a much lower relative abundance in the gut of new- 86 borns (16). The subdominant butyrogenic species could however quickly become 87 more dominant upon weaning as a result of the introduction of solid food and the 88 cessation of breast-feeding (2, 17). The colonisation by the strict anaerobic, butyrate- 89 producing bacteria could be a critical step for the gut and immune maturation (18, 19). 90 The interactions between lactate-producing bacteria (such as bifidobacteria) and 91 lactate-utilising bacteria (such as Ruminococcaceae and Lachnospiraceae) are 92 suggested to be associated with a lower risk of colicky symptoms and atopic disease 93 in infants (18-21). To date, cross-feeding between glycan-degrading bifidobacteria 94 and butyrate-producers using complex dietary carbohydrates (including starch, inulin, 95 fructo-oligosaccharides, and arabinoxylan oligosaccharides) has been demonstrated 96 in in vitro co-culturing experiments (22-26). However, limited studies have shown the 97 cross-feeding between these groups of bacteria on host-secreted glycans such as 98 HMOS (27) and mucins (28). 99 In this study, we investigated the trophic interaction between an HMOS- 100 degrader, Bifidobacterium longum subsp. infantis and a butyrogenic non-degrader of 101 human milk carbohydrates. To this end the butyrate-producer Anaerostipes caccae 102 was used as the representative species for the Lachnospiraceae family as it is 103 detected in the early life gut microbiota (2, 29) and is one of the prevalent members 104 of the gut microbiota in human adults (30). We show that B. inf supports the 105 development of the microbial ecosystem by metabolising lactose and HMOS into 106 monosaccharides and short chain fatty acid (SCFA) including lactate and acetate, to 5 107 support the growth and concomitant butyrate production by A. cac. This butyrogenic 108 cross-feeding demonstrates the importance of bifidobacteria in the establishment of a 109 healthy microbial ecosystem in early life. 110 111 Results 112 The occurrence of B. inf and A. cac across the life span 113 A published dataset (29) was mined for the occurrence of B. inf and A. cac in the 114 microbiota across life stages. The two infant-associated bacteria demonstrated 115 opposite trajectories in early life. Bifidobacterium genus showed high abundance at 116 the first year followed by a sharp decline, with a negative correlation between age 117 and relative abundance (Spearman ρ = -0.38, p < 0.05) (Fig. 1). On the contrary, 118 Anaerostipes genus (Spearman ρ = 0.56, p < 0.05) and Lachnospiraceae family 119 (Spearman ρ = 0.37, p < 0.05) were present at low abundance early in life and 120 increased in relative abundance in the first 1000 days of life (Fig. 1). 121 122 Model for B. inf and A. cac co-occurrence 123 Bacterial strains were cultured in anaerobic bioreactors controlled at pH 6.5 and 124 supplemented with either lactose or total human milk (HM) carbohydrates. B. inf 125 monoculture reached maximal cell density around 12 h (ODmax = 1.40 ± 0.38 in 126 lactose and ODmax = 1.37 ± 0.25 in total HM carbohydrates) (Fig. 2). For A. cac 127 monoculture, no growth or substrate degradation was detected in identical media 128 (ODmax = 0.02 ± 0.01 in lactose and ODmax = 0.03 ± 0.02 in total HM carbohydrates) 129 (Table S1). The co-culture of B. inf with A. cac grew rapidly reaching maximal optical 6 130 density at 11 h in lactose (ODmax = 3.63 ± 0.61) and at 9 h in total HM carbohydrates 131 (ODmax = 3.54 ± 0.60). The community dynamic in the co-cultures was monitored 132 over time by qPCR. An equal amount of B. inf and A. cac (around 106 copy 133 number/ml) was inoculated at the start of the fermentation. During the first 7 h, B. inf 134 and A. cac increased 100-fold based on the increase of 16S rRNA gene copy 135 number, after which growth slowed down. FISH analysis of samples harvested at 11 136 h showed B. inf to A. cac ratio of 1:6. This was observed for both conditions either in 137 lactose or total HM carbohydrates supplemented cultures. 138 139 B. inf supported the growth and metabolism of A. cac in lactose and HMOS 140 The substrate consumption and SCFA production were monitored over time (Fig.