Effective establishment of donor gut microbiota in gnotobiotic mice


 Background: Determining whether associations between gut microbiota characteristics and host physiology represent causal relationships is a fundamental challenge for microbiome research. One way these associations can be investigated is to instil donor faecal material into gnotobiotic mice and to assess the extent to which donor phenotype is recapitulated. However, the manner in which this process is performed varies considerably between studies, and assessment of microbiota re-establishment in recipient animals is not always carried out. We report a detailed investigation of microbiome assembly in germ-free mice and compare the effects of single and multiple rounds of faecal gavage, using both native and antibiotic-disrupted donor material. Results: Levels of bacteria within the faeces of recipient animals increased rapidly following the instillation of donor material. However, considerable instability in microbiota composition continued during the first two weeks post-gavage, with substantial changes in taxon relative abundance occurring in parallel to declining faecal pH. Relative compositional stability was not achieved until day 28 and persistent differences between recipient and donor microbiota remained. These included an increased relative abundance of Bacteroidetes, and a reduced relative abundance of Firmicutes. Of taxa detected in donor material, 52% were represented in stable recipient microbiota following transplantation with native faecal material (single gavage), increasing to 66% following three rounds of gavage. These taxa accounted for 95% and 91% of total donor bacterial abundance, respectively. Performing multiple rounds of gavage significantly increased microbiota similarity between donor and recipient, and significantly reduced within-group dispersion (P<0.05). Instillation of antibiotic-associated microbiota resulted in substantially lower temporal and inter-animal variance, with multiple rounds of gavage providing no substantial benefit. Conclusions: Microbiome assembly in recipient animals is not immediate and several weeks are required for microbiota stability to be achieved. Multiple rounds of faecal gavage result in greater similarity to donor microbiota and reduced inter-animal variance. The process of donor microbiota re-establishment, and therefore the interval required prior to investigations using recipient animals, is influenced by donor microbiota characteristics.


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Introduction 48 Much of our understanding of relationships between the gut microbiome and human 49 physiology comes from observational studies. Having identified associations between gut 50 microbiome traits and host measures, the next challenge is to determine whether these 51 relationships are causal, secondary to observed physiological phenomena, or result from 52 parallel but independent processes. 53 One of the few experimental strategies available to assess causality in host-microbiome 54 interactions is the instillation of intestinal microbiota from human donors or animal models 55 into germ-free mice, and assessment of whether donor phenotype is recapitulated. This 56 approach has been used, for example, to demonstrate that gut microbiota contributes to obesity 57 in the context of a high fat diet [31], to underdevelopment in the context of a low nutrient 58 density/bioavailability diet [4], and to variation in treatment efficacy in recipients of cancer 59 immunotherapy [18,34]. The employment of this strategy is becoming increasingly popular 60 due to growing access to gnotobiotic facilities, and evidence that antibiotic depletion of 61 recipient intestinal microbiota in conventional mice prior to faecal transfer represents a 62 relatively poor alternative [13,37]. 63 Despite the utility of gut microbiota transplantation as a means to understand microbiome-host 64 relationships, there is little consistency in the manner in which the technique is performed. In 65 particular, the number of rounds of gavage employed varies between studies, as does the period 66 allowed to elapse between the final gavage and the initiation of the experiment or assessment. 67 The extent to which donor microbiota are replicated within recipient animals is commonly 68 neglected, and where assessment is performed, it often focuses donor taxon presence/absence, 69 rather than microbiota structure or composition. 70 Many of the bacterial clades that are closely associated with the regulation of host physiology 71 are obligate anaerobes [33,46], and are particularly susceptible to loss of viability during the 72 processing of material for transplant [26,27]. Not only can failure to establish such taxa in the 73 gut lumen of gnotobiotic recipient animals lead to divergence in microbiota composition from 74 donor animals, but it can allow the proliferation of opportunistic facultative anaerobes [22,42]. 75 Such changes can have profound implications for the metabolic and immuno-regulatory 76 properties of the gut microbiome [1,16]. 77 A number of previous studies have aimed to describe the process of intestinal microbiota 78 assembly in germ-free mice [12,17]. In particular, Gillilland and colleagues described 79 microbiota assembly at two mucosal sites, the caecum and the jejunum, during the first 21 days  87 We investigated the dynamics of donor microbiome assembly in the gut of recipient germ-free 88 mice, including a comparison of the effects of single and multiple rounds of gavage. 89 Assessment of microbiota transplantation was performed using both native and antibiotic-90 disrupted gut microbiota, with the latter used to represent changes that are commonly described 91 in association with pathophysiology [25,39].  95 In mice that received native donor microbiota (approximately inoculum of 10 5 bacterial cells), 96 total bacterial load peaked between four days post initial gavage (D4) and seven days post 97 initial gavage (D7), before declining to D14 ( Figure 1A). From D14, faecal bacterial load was 98 broadly stable at approximately 5x10 6 bacterial cells/mg. No significant differences were 99 observed between mice that received one or three rounds of gavage.  single and multiple rounds of gavage remained significant throughout the 70-day study 118 (median, IQR: 1G= 33.3%, 33.3-37.5; 3G= 37.5%, 37.5-39.6; P < 0.05, except at D42).

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The percentage of donor taxa detected in recipient animals fluctuated over the 70-day period 120 of assessment. As mice were housed in a controlled environment in which the only route of  145 Recipient-donor microbiota similarity was assessed based on weighted UniFrac distance.

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Similarity increased in both groups to D35, after which no significant differences were  Microbiota composition was also assessed based on distance to group centroid. In recipients of 161 native microbiota, distance to centroid increased from D4, peaking at D7, and declining 162 thereafter ( Figure 5A). Distance to centroid was higher at all but one time-point (D49) in mice 163 that received one round of faecal gavage compared to those that received three, achieving 164 statistical significance at D14, D28, and D63 (Mann-Whitney test, P< 0.05). Distance to group 165 9 centroid in recipients of antibiotic-disrupted microbiota did not differ significantly between 166 single and multiple gavage groups ( Figure 5B).

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Temporal dynamics in taxon relative abundance 169 Temporal changes in taxon relative abundance differed substantially between phylogenetic  Bacteroidales, and Verrucomicrobiales) were broadly stable, although a sustained increase in 183 Bacteroidales was observed between D28 and D35. In contrast to recipients of native donor 184 microbiota, the relative abundance of Ruminococcaceae fell substantially following instillation 185 until D7, before partially recovering. Again, no significant differences were observed between 186 single and multiple gavage groups.

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At the phylum level, substantial differences between donor and recipient microbiota were 188 evident at D7, persisting to D70 ( Figure S4). In particular, the relative abundance of the most  we observed the total bacterial levels in recipient animals to resemble donor animals rapidly.

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The rate of microbiota expansion was not affected by the number of rounds of gavage 219 performed. Increases in bacterial diversity during the early stages of gut colonisation are 220 constrained by ecological succession rather than by rate of biomass increase, as described in 221 vaginally-born human infants [43]. Succession involves a process whereby early gut colonisers

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The changes in microbiota characteristics that we observed following instillation into germ-237 free mice were consistent with ecological succession. In mice transplanted with native 238 microbiota, dissimilarity to donor microbiota and within-group dispersion were highest during 239 the initial two weeks period, with changes in keystone bacterial clades consistent with well-  In addition to investigating microbiota assembly in mice transplanted with native microbiota, 276 we also assessed colonisation dynamics following the instillation of microbiota from antibiotic-277 exposed animals. In doing so, our aim was to determine whether the dynamics of  In our study, caecal microbiota were harvested and processed under strict anaerobic conditions.

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Alternative approaches that utilise stool, or that involve exposure to aerobic conditions, are 298 likely to be associated with lower levels of donor-recipient similarity. Furthermore, our 299 transplantation was between members of the same species, subject to similar environmental 300 and dietary exposures. A growing number of studies involve the instillation of human stool, or 301 stool-derived microbiota, into gnotobiotic mice. Substantial differences in genetics, 302 physiology, anatomy, and diet, would be expected to further reduce associated levels of 303 microbiota recapitulation.

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Our study had a number of limitations. For example, we did not attempt to assess all possible