B. theta VPI-5482 can phase vary the expression of eight different capsular polysaccharides (WT). B. theta Δcps strain that cannot produce capsule was generated previously by sequentially deleting all CPS gene clusters (acapsular) (Porter et al., 2017). We first established neutrally tagged clones of these strains by inserting a genetic barcode linked to an antibiotic resistance cassette and a fluorescent protein gene into the genome using the previously described pNBU2 integration plasmid. Six barcode sequences, previously developed and validated for Salmonella (wild-type isogenic tagged strains [WITS]; Grant et al., 2008; Maier et al., 2014) were inserted adjacent to an erythromycin-resistance cassette, ermG (Cheng et al., 2000), and a GFP or mCherry fluorescent protein gene under a strong constitutive promoter. Untagged strains were generated by inserting pNBU2 carrying the tetQb tetracycline resistance cassette (Nikolich et al., 1992) and a GFP gene under the control of a strong constitutive promoter into the same integration site (Figure 1A, Key Resources Table). Fluorescent proteins (GFP or mCherry), expressed from a phage promoter with an optimized ribosome binding site, were included to permit later visualization of clones (Wang et al., 2000; Whitaker et al., 2017).

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Barcoded B. theta strains have similar fitness for growing in vitro and in vivo.

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We validated a system to enrich barcoded strains from overall very low frequencies (Figure 1B) Samples were plated on BHIS agar with gentamycin to determine the total B. theta CFU and on BHIS agar containing the appropriate antibiotic (erythromycin or tetracycline) to determine the total barcoded B. theta CFU. Subsequently, all B. theta was washed from the plates, and genomic DNA extracted. The relative frequency of each barcode among the recovered colonies was obtained by qPCR using primer sets specific for each barcode. CFU of each individual barcoded strain can then be determined by simple multiplication. Serial dilution and recovery of barcoded strains in in vitro systems shows excellent resolution over five orders of magnitude (Figure 1—figure supplement 1).

A critical assumption of any analysis using genetically barcoded strains is that the chromosomal insertions, as well as the construction process, have not altered the fitness of the strains compared to the WT strain. Anaerobic growth in BHIS media was near-identical in all the barcoded B. theta strains (median doubling time ranged from 78 to 90 min) (Figure 1C). Correspondingly, the barcoded strains maintained their relative abundances, as evaluated by qPCR, when all barcode strains were mixed and grown overnight (Figure 1—figure supplement 2A and B). Whole-genome sequencing of the barcoded strains revealed a few synonymous and non-synonymous mutations, as would be expected for the construction process of individual strains (Supplementary file 1). However, none of the identified mutations is expected to have a major fitness effect, consistent with our observed data.

To test whether the tags confer a fitness effect upon colonization of a host, we colonized germ-free (GF) mice with a uniform mixture of 10⁶ CFUs of the untagged strain and each barcoded strain (Figure 1D). At this barcode abundance, stochastic loss of tags is highly unlikely. We compared the relative abundance of each barcode in the inoculum to that in the cecal content and feces after 48 hr of colonization. These experiments revealed small, random deviations in the distribution, consistent with uniform fitness (Figure 1E). Finally, as erythromycin- and tetracycline resistance were used to distinguish the barcoded and untagged B. theta strains, we also tested whether the antibiotic resistant cassettes alter competitive fitness. In culture, we found a minor competitive advantage of the tetracycline expressing strains over erythromycin only in the acapsular strain (Figure 1—figure supplement 2C). However, there was no significant competitive advantage of any antibiotic cassette after 2 days of colonization in GF mice (Figure 1F). As all barcoded strains carried the erythromycin resistance cassette, small fitness effects associated with the cassette will not affect competition between the barcoded strains. In competitive colonization, tetracycline and erythromycin resistances were reversed in two sets of experiments with similar results, and a simple model based on this data suggests a maximum error due to the antibiotic fitness effect of twofold. This is small compared to the relative competitive fitness of the acapsular and wildtype B. theta strains. Therefore, while absolute equivalent fitness is near-impossible to achieve in these systems, the error due to unintended fitness effects is within an acceptable range.

We then applied the neutrally barcoded B. theta strains to estimate colonization probabilities. As invasion probabilities depend on the interaction with the resident microbiota (Kurkjian et al., 2021), we probed B. theta colonization in mice carrying three different communities: low-complexity microbiota (LCM) Stecher et al., 2010; Oligo Mouse Microbiota (OligoMM12) (Brugiroux et al., 2016), and SPF microbiota. These include two low-complexity microbiota models with a reduced set of strains (LCM: 8 strains and OligoMM12: 12 strains) and the closest model for complete microbiota in laboratory mice (SPF: 12 families that include several species) (Figure 2—figure supplement 1). We evaluate the distribution of barcoded B. theta cells in cecum content at 48 hr after initial colonization, a time point shortly after the B. theta population in the cecum reaches carrying capacity (Figure 2—figure supplement 2).

Assuming that the change in relative abundance of tags before and after the colonization process is due to stochastic loss of B. theta, we formulated a simple ‘initial’ model that allows us to infer a per-cell colonization probability for B. theta. The model assumes that B. theta cells undergo random killing during their transition through the stomach and small intestine, that is, the population experiences an initial bottleneck event. Surviving cells arriving in the cecum start growing and the clonal progeny of these cells can be quantified at 48 hr post-colonization via a combination of plating and qPCR (Figure 2A). The number of cells of an individual barcoded clone in the inoculum, n₀, is low in our experiments. Correspondingly, the distribution of barcoded bacteria introduced into the stomach of each animal is better approximated by a Poisson distribution of mean n₀ than by a uniform distribution. The probability of losing a barcoded clone can be considered equivalent to the fraction of barcoded clones lost across all animals. Considering this early loss of clones as a binomial sampling process, we can express this probability as $e^{-\beta n_0}$ , where β is the colonization probability of an individual clone from the inoculum. β can therefore be simply computed for animals all receiving an identical inoculum. To increase the power of our observations, we have also pooled data across multiple experiments with small deviations in n₀ by maximizing the likelihood of the experimental observations (Figure 2—figure supplements 3 and 4). A more complex calculation can be carried out using the variance of the barcoded population rather than defined loss/retention, a method that can take more information into account, although it is more complicated to execute (see ‘Mathematical modeling’).

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Colonization probability of B. theta strains in low-complexity microbiota (LCM), Oligo Mouse Microbiota (Oligo-MM12), and specific pathogen free (SPF) mice.

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It is important to note that if all tags are lost, or if all tags are recovered, only upper or lower bounds for β can be estimated. Correspondingly, experiments where some, but not all, tags are lost from the final population yield maximum information. To find the optimal n₀ that leads to partial barcode loss in vivo, we titrated the barcoded strains into an untagged B. theta population to give n₀ values ranging from 18 to 26,666 CFU for each barcoded strain (Figure 2—figure supplements 3 and 4). This was carried out in the context of three different microbiota communities, and for both WT and acapsular B. theta. Recovery of total B. theta and CFUg^–1 of barcoded strains from the cecum was determined at 48 hr post-colonization. We used the Pielou evenness (Pielou, 1967) as a summary representation of the distributions of barcoded population (Figure 2B, Figure 2—figure supplements 3 and 4). The resulting β estimates are most robust at an n₀ which results in approximately half of the tags being lost (see Appendix 1 ‘Supplementary methods’). In the case of LCM and OligoMM12 low-complexity microbiota mice, an n₀ of between 10 and 100 was optimal for both wild-type and acapsular B. theta, that is, 10–100 CFU of each barcoded strain was spiked into an inoculum of 10⁷ untagged B. theta. In the SPF mice, carrying a complex microbiota, an n₀ of around 500 was informative for WT, but 5000 CFU were needed of each acapsular barcoded strain.

Finally, we challenged the assumption that loss of barcoded clones was due to stochastic loss. Selective sweeps of a clone, or clones, that have acquired a beneficial mutation would also explain barcode loss. We therefore reisolated abundant barcoded B. theta strains from the 48 hr time point from previous experiments. These were used to assemble an inoculum in which some barcodes were represented by re-isolated strains and others by original ancestral strains. These were mixed at a high n₀ (approximately 2 × 10⁶ CFU of each clone per inoculum) and used to colonize SPF mice. There was no consistent advantage of re-isolated strains over ancestral strains in colonizing the cecum at 48 hr post-colonization (Figure 2—figure supplement 5A), consistent with the absence of strongly beneficial mutations in surviving clones. To further confirm the assumption of equal stochastic loss of each barcode, we re-calculated β for all experiments excluding each individual barcode in turn. Excluding data from any one barcode has no statistically significant effect on the estimates of β, as calculated using barcode loss (Figure 2—figure supplement 5B) or variance (Figure 2—figure supplement 5C).

The resident microbiota composition in the mammalian gut is one of the main factors constraining the colonization of newly arriving species. This can happen through various mechanisms such as competition for nutrients (Bäumler and Sperandio, 2016; Maier et al., 2013), modification of the intestinal environment (Cremer et al., 2017), via direct suppression of the invaders by phages (Almeida et al., 2019; Barr et al., 2013) or type VI secretion systems (Chatzidaki-Livanis et al., 2016; Wexler et al., 2016). Additionally, the microbiota stimulates host mucosal immunity and influences intestinal physiology (Zheng et al., 2020). B. theta loads in the cecal content at 48 hr post-inoculation were similar in LCM and OligoMM12 mice, but significantly lower in SPF mice (Figure 2C). The colonization probability, β, of barcoded B. theta WT strains, calculated using loss or variance methods, was also lower in SPF mice (Log10(β, colonization probability) ± 2 standard deviations = –2.35 ± 0.14) compared to the two LCM (–1.50 ± 0.10; and Oligo, –1.54 ± 0.13) (Figure 2D, Figure 2—figure supplement 6A). Of note, while the relative size of the final population is 100-fold lower in SPF mice, and the relative colonization probability is only 10-fold lower than in animals with a low-complexity microbiota. This indicates that size of the open niche does not linearly translate into colonization probability, that is, the neutral tagging approach reveals information that cannot be simply gleaned from standard CFU determination.

As CPS are thought to play a crucial role in phage evasion/infection (Porter et al., 2020), immune evasion (Fanning et al., 2012; Hsieh et al., 2020; Porter et al., 2017), and protection from other environmental stressors, we expected to see a decreased colonization probability for acapsular B. theta strains in all microbiota backgrounds. Surprisingly, in LCM mice, the total population size of acapsular B. theta (Figure 2E) and the probability to colonize (Log10β: LCM, –1.43 ± 0.13; and OligoMM12, –1.49 ± 0.14) were not significantly different to the WT strain (Figure 2F, Figure 2—figure supplement 6B). There was therefore no measurable fitness benefit of CPS in gut colonization up to 48 hr post-inoculation in these settings. However, we observed a different scenario when we inoculated acapsular B. theta into mice carrying a SPF microbiota. Both the total population size of acapsular B. theta (Figure 2E) and the colonization probability (Log10β: SPF, –3.65 ± 0.13; Figure 2F) were tenfold lower compared to the WT strain, indicating a strong fitness benefit of CPS in the context of a more diverse microbiota. We could not definitively tie this increased clearance to any particular host or microbial mechanism: SPF mice do not have measurable IgA titers specific for acapsular B. theta (Figure 2—figure supplement 7A), nor could we identify lytic spots produced by phage specific for acapsular B. theta from the cecum content of SPF mice (Figure 2—figure supplement 7B). As microbiota-driven restriction of acapsular B. theta colonization is expected to establish very rapidly on recolonization of a GF mouse, but host-driven mechanisms such as upregulation of antibody responses may take several days to weeks, we compared the acapsular B. theta colonization probability in ex-GF mice that had been recolonized by rehousing with SPF mice for 2 days or for 2 weeks. Although short-term recolonization resulted in a larger B. theta population in the cecum than long-term re-colonization with an SPF microbiota (Figure 2—figure supplement 8A), the colonization probability was near-identical between the two groups (Figure 2—figure supplement 8B). Therefore, mechanisms restricting colonization of acapsular B. theta in SPF mice are either direct microbial competition (e.g., via type VI secretion) and/or are very rapidly induced host mechanisms, or a combination of both.

The absence of a decreased colonization probability for acapsular B. theta in LCM mice apparently conflicts with previous studies showing a competitive fitness defect of this strain (Coyne et al., 2008; Porter et al., 2017). We therefore carried out competitive colonizations with B. theta WT and acapsular strains in all microbiota backgrounds. Starting at a 1:1 ratio, we inoculated mice with erythromycin-resistant WT and tetracycline-resistant acapsular B. theta and quantified the cecal bacterial load 48 hr post-inoculation (Figure 3—figure supplement 1A). This reveals a gradient of competition with the WT winning over the acapsular strain, obtaining a competitive index (abundance of WT over acapsular B. theta at the end of the experiment) of approximately 20 in GF mice, 100 in LCM mice, and 10⁴ in SPF mice (Figure 3A). Therefore, the competitive fitness benefit of CPS increased in proportion to microbiota complexity, despite the fact that no difference on colonization probability of the WT and acapsular B. theta could be detected on single colonization.

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Competitive colonization with acapsular and WT strains.

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To better understand the mechanisms generating a competitive disadvantage for the acapsular strains, we performed a competition experiment in the same microbiota backgrounds, but this time, using tetracycline-resistant B. theta WT and barcoded erythromycin-resistant acapsular B. theta strains. WT strain density (Figure 3—figure supplement 1B) and the average increase in the WT relative to the acapsular strains (after adjusting for the initial ratio in the inoculum, Figure 3—figure supplement 1C) were similar between the two experiments. Interestingly, the colonization probability β was lower for the acapsular B. theta strain when co-colonizing with the WT strain than when colonizing alone (Figure 3B). This indicates that the competition with WT B. theta results in both a lower total population size and increased clonal extinction in the acapsular strains.

To understand how acapsular B. theta can have an indistinguishable colonization probability when inoculated alone, but a major fitness defect in competition with B. theta WT, we carried out time courses of feces collection to estimate the net growth rates (i.e., growth minus clearance) of both strains colonized individually in OligoMM12 mice (Figure 3—figure supplement 2). Using peak-to-trough analysis, we observed that growth rates of B. theta in feces and cecum is similar, and higher than all other OligoMM12 strains at 8 hr post-inoculation, as would be predicted for active growth (Figure 3—figure supplement 3). Longitudinal feces collection was therefore used as a proxy for large-intestinal colonization. Tracking B. theta CFUg^–1 in feces over 48 hr demonstrated that the net growth rate of acapsular B. theta is lower than that of WT (Figure 3C, WT: 0.50/hr and acapsular: 0.40/h, p<0.01). Interestingly, detectable exponential growth of acapsular strain starts around 4.5 hr later that for the WT (Figure 3D, WT: 3.2 hr and acapsular: 7.7 hr, p=0.02). As there is an inherent detection limit for CFU, as well as an intrinsic time delay due flow through the gastrointestinal tract this delay could be explained by (1) a classic lag phase (i.e., period of adaption before growth begins), (2) strongly increased killing of the acapsular B. theta during stomach and small intestinal transit, or (3) retention of acapsular B. theta in the non-growth-permissive small intestine. We could exclude differential retention of acapsular B. theta in the small intestine. Analysis of B. theta distribution in the small and large intestine at 8 hr post-colonization indicated that most B. theta had already arrived in the large intestine at this time point. There was no evidence of differential retention of acapsular and WT B. theta in the small intestine (Figure 3—figure supplement 4). We can also largely exclude killing prior to reaching the cecum as B. theta WT and acapsular clones have a very similar probability of colonization in single colonizations of OligoMM12 mice (Figure 2 and figure supplements). For early killing to explain out-competition of the acapsular strain, it would be necessary that the presence of WT B. theta increases acapsular B. theta killing in the stomach or small intestine. As the bacteria are at low density during early infection, it is unlikely that they affect either each other or the host prior to arrival in the large intestine. In contrast, the delay in detectable growth of the acapsular strain is observed both competitive and single colonization of OligoMM12 mice, consistent with a classical lag phase. The magnitude of the delay to detection is similar on single and competitive colonization with WT B. theta (Figure 3—figure supplement 2E and F), consistent with this being an intrinsic feature of the acapsular B. theta strain. We therefore propose that acapsular B. theta exhibits a classical extended lag phase in vivo, likely due to a longer adaption period for the acapsular B. theta to growth in the gut environment.

To further explore this hypothesis, we extended our one-step colonization model to include both a difference in lag phase after arrival in the cecum and/or a difference in net growth rates (Figure 3E). Combining these additional variables generated a model that quite well recapitulates the expected competitive fitness (Figure 3F) and colonization probabilities (Figure 3G) (see 'Methods' for a brief description of the model and Appendix 1 'Supplementary methods' for the description of all parameters used). Running the same model based only on identical growth rates, but different lag phase produces a worse prediction of the competitive index, while omitting the lag-phase difference produces a similarly good fit (Figure 3—figure supplement 5). Therefore, the competitive fitness defect of the acapsular B. theta strains can be explained by a slightly slowed in vivo net growth rate, with a small contribution from an extended in vivo lag phase.

Finally, as a proof of concept for neutral tagging in the study of established microbiota, we used our system to probe clonal extinction when an established B. theta population in the gut is challenged by two major environmental perturbations: (1) shifting from standard chow to a high-fat no-fiber diet (HFD) (David et al., 2014; Wotzka et al., 2019) and (2) infectious inflammation driven by Salmonella Typhimurium (Stm) (Maier et al., 2014). To exclude microbe–microbe interactions from the possible observed mechanisms, we monocolonized GF mice with a mixture of untagged and barcoded B. theta WT strains such that all tags were present with a roughly uniform distribution prior to intervention, that is, minimum loss, in the gut lumen prior to challenge (Figure 4A, Figure 4—figure supplement 1).

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Acute challenges imposed a population bottleneck in the resident B. theta population.

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In the first set of challenges, after 4 days of colonization, we exposed mice to oral infection with 10⁶–10⁷ CFUs of a Stm strain either attenuated (SL1344 ΔssaV, no SPI-2) or fully avirulent (SL1344 ΔinvGΔsseD, no SPI-1 or SPI-2). Despite similar Stm loads (Figure 4—figure supplement 2A), the attenuated strain induces moderate intestinal inflammation in GF mice while the fully avirulent strain does not induce visible gut inflammation (Hapfelmeier et al., 2005; Stecher et al., 2005; Figure 4—figure supplement 2B). B. theta populations were monitored in feces before the challenge was administered and 3 days after the challenge in feces and cecum. In line with the models presented above, cecum content values were used for inference of the bottleneck size.

After 3 days of infection with attenuated Stm, the total B. theta population was reduced approximately 100-fold in feces (Figure 4B). In addition, the probability of an established clone to survive this acute inflammation challenge was approximately 1 in 2000 in cecum (Figure 4C). This means that the initial estimated population of 10¹⁰ CFUg^–1B. theta in the cecum is reduced to an effective population of between 10⁶–10⁷ clones, while maintaining a total population size of between 10⁹–10¹⁰ CFUg^–1 (Figure 4B). Challenge with avirulent Stm has a limited effect on total population density of B. theta (Figure 4B); however, it still induces a bottleneck of approximately 1 in 250 in the cecum, potentially due to subclinical inflammation induced by colonization with a noninvasive Enterobacteriaceae (Figure 4C). Of note, we cannot formally exclude direct toxicity of Salmonella against B. theta based on these data. Rather around 90% of the B. theta clearance observed in virulent Salmonella infections can be attributed to the strong inflammatory response induced (Figure 4—figure supplement 2B).

In a second set of experiments, B. theta monocolonized mice were fed an HFD. Despite setting the initial barcoded population size in the cecum at between 100 and 1000 CFUg^–1 (Figure 4—figure supplement 1), HFD feeding did not significantly change the cecum population size (Figure 4B), nor did it increase the loss of barcoded B. theta (Figure 4C). To calculate our limit of sensitivity with this system, we estimated that detecting loss of one barcode in one of the tested mice would occur with a bottleneck population size of between 10⁹ and 10¹⁰ clones, that is., a population contraction of up to tenfold would be within the experimental noise of our measurements. Published reports indicate that B. theta is sensitive to bile acids (Wotzka et al., 2019), which should be abundantly induced by HFD feeding. The conclusion of the data is therefore rather than the current neutral tagging system is not sufficiently precise to detect the magnitude of population dynamics changes induced by HFD consumption in GF mice.

B. theta strains were grown anaerobically (5% H₂, 10% CO₂, rest N₂) at 37°C, overnight in brain heart infusion (BHI) supplemented media (BHIS: 37 g/L BHI [Sigma]; 1 g/L-cysteine [Sigma]; 1 mg/L Hemin [Sigma]). For enrichment cultures in plates, we used BHI-blood agar plates (37 g/L BHI [Sigma]; 1 g/L-cysteine [Sigma]; 10% v/v defribinated sheep blood [Sigma]). Antibiotics were added to liquid cultures or plates as required for strain selection: erythromycin 25 µg/L or tetracycline 2 µg/L. In the case of BHI-blood agar plates used for cloning or gut content enrichment, we additionally added gentamycin 200 µg/L to all plates to prevent growth of other microbiota species. Plates were incubated for 48–72 hr at 37°C in anaerobic conditions. For a complete list of the bacterial strains used in this study, see Key Resources Table.

Genetic tags, fluorescent proteins, and antibiotic resistance were introduced by using the mobilizable Bacteroides element NBU2, which integrates into the Bacteroides genomes at a conserved location at either BTt70 or BTt71 (Wang et al., 2000). Gene fragments containing a unique 40 bp sequence (biding site for forward primer) and a 609 bp sequence with the ydgA pseudogene (common binding site for reverse primer) were synthesized (gBlocks, Integrated DNA Technologies) and cloned by Gibson Assembly Master Mix (NEB) into an NBU2 plasmid carrying the erythromycin-resistant cassette ermG (barcoded B. theta strains) and a fluorescent GFP or mCherry protein (see Key Resources Table for specific combination of fluorescent protein and tag). A similar NBU2 plasmid carrying the tetracycline-resistant cassette tetQb and the GFP protein was used to construct the untagged strains (B. theta untagged). All fluorescent protein genes have high-expression promoter and an optimized RBS (Whitaker et al., 2017). For both, we used 10 µL of desalted assembly reaction products to transform competent E. coli S17-1 cells (mid-log cells, washed three times in deionized ice-cold water) by electroporation (V = 1.8 kV; MicroPulser, Bio-Rad). After 1 hr recovery at 37°C in 1 mL of LB, cells were plated on LB plates with chloramphenicol (12 µg/mL) and grown overnight. Plasmid-carrying E. coli S17-1 and B. theta strains were cultured overnight in 5 mL of liquid media. E. coli S17-1 and B. theta were washed with PBS, pooled in 1 mL of PBS, plated BHI-blood agar plates without antibiotics, and grown aerobically at 37°C for at least 16 hr. The lawn of E. coli S17-1 and B. theta was collected in 5 mL of PBS, homogenized by vortex, and 100 µL were plated in BHI-blood agar plates supplemented with erythromycin 25 µg/L and gentamycin 200 µg/L. After 48 hr, single colonies were streaked in fresh BHI-blood agar plates with antibiotics to avoid potential contamination with WT strains. Successful insertion in the BTt70 or BTt71 sites was evaluated by PCR (Key Resources Table). To minimize potential variation, we used strains with a single insertion in BTt70. In summary, the barcoded strains carried the barcode, erythromycin resistance cassette and fluorescent protein (GFP or mCherry) in the genome. The untagged B. theta carried a tetracycline resistance cassette together with a GFP protein inserted at the same position in the genome.

One investigator blinded to sample metadata (i.e., microbiota, bacteria strain used, acute challenge used) performed the sample processing and qPCR quantification of barcodes. Samples were serially diluted and plated on appropriate selective BHIS agar (gentamycin 200 µg/L plus either erythromycin 25 µg/L or tetracycline 2 µg/L) and cultured in 5% H₂, 10% CO₂, rest N₂, at 37°C for 48 hr (Coy Anaerobic tent). CFU determination was carried out by counting, then colonies were washed from the plates (all plates with at least 30 colonies were included), pooled in 5 mL of PBS and homogenized by vortex. Genomic DNA was isolated with the QIAamp DNA Mini Kit (QIAGEN). qPCR was performed using with FastStart Universal SYBR Green Master Mix (Roche, Cat# 4385610). Primers (Key Resources Table) were mixed to a final concentration of 1 µM. Between 160 and 200 ng of DNA was amplified in duplicates using StepOne Plus or QuantStudio 7 Flex instruments (Applied Biosystems) using the following protocol: initial denaturation at 95°C for 14 min followed by 40 cycles of 94°C for 15 s, 61°C for 30 s, and 72°C for 20 s as described previously. As qPCR reactions for these barcodes have identical efficiencies, genomic DNA extracted from a single barcoded strain was used as an internal standard and CT values were backcalculated to this standard curve to generate a relative frequency of each barcode in the pooled colonies. These relative frequencies were then multiplied by the total CFU/g of barcoded B. theta to obtain the CFU/g of each barcoded strain.

Individual B. theta strains were grown overnight on BHIS. Stationary-phase cultures were washed with PBS, and O.D. was quantified and adjusted to 0.05 in 200 µL of fresh BHIS in a round 96-well tissue culture plates. Plates were transferred into the anaerobic tent and growth was quantified at 37°C with shaking using a plate reader (Infinite PRO 200, Tecan).

For competition experiments, stationary-phase cultures B. theta WT or acapsular strains were washed with PBS, O.D. was quantified and adjusted to approximately 5 × 106 CFU/mL per strain (one B. theta untagged and six B. theta barcoded strains) in 10 mL of fresh BHIS. An aliquot of this mix was serially diluted and plated in BHI-blood agar plates with the respective antibiotics for CFU quantification. Cultures were kept overnight, with shaking (800 rpm) at 37°C in the anaerobic tent. Afterward, an aliquot was plated as described before. For assessing the competition between B. theta barcoded strains, we isolated DNA from one of the dilutions used for quantification and assessed the relative distribution of the tags by qPCR (see ‘Quantitative PCR’ section). For assessing the competition among strains with different antibiotic resistances, we calculated the competition index by dividing the CFU/mL of the untagged B. theta untagged strain (tetracycline-resistant) by the adjusted number of B. theta barcoded strains (erythromycin-resistant; CFU/mL divided by six, as all the barcoded strains were present in the culture).

All animal experiments were performed with approval from the Zürich Cantonal Authority under license number ZH120/19 and ZH009/21. In all experiments, we used mice with C57BL/6J genetic background, between 12 and 15 weeks old and of variable gender. C57BL/6J GF and gnotobiotic mouse lines (LCM [Stecher et al., 2010]; OligoMM12 [Brugiroux et al., 2016]) were raised in surgical isolators under high hygiene standards at the ETH Phenomic Center and were regularly tested for contamination by aerobic and anaerobic cultivation, culture-independent assessment of intestinal bacterial densities and serology/PCR for common viruses and eukaryotic pathogens. Note that all LCM mouse lines were bred for 1 year (OligoMM12) or more than 10 years (LCM) with their gnotobiotic microbiota. C57BL/6J SPF mouse line was raised in IVC cages in a different barrier unit of the same facility. Mice were transferred to our experimental facility in sterile, tight closed cages and house into the IsoCage P- Bioexclusion System (Tecniplast) for 24–48 hr before the experiment to adapt to new housing conditions. In all experiments, standard chow and water was prepared under strict aseptic conditions to avoid any potential contaminations. Although mice themselves were not randomized on each experiment, cages containing appropriate mouse numbers were randomly assigned to each inoculum/treatment and both genders are represented in all groups.

B. theta WT WITS 01 or B. theta acapsular WITS 01 strain was grown overnight in BHIS. Stationary-phase cultures were washed with PBS, and an inoculum of ~5 × 10⁷ CFUs/100 µL dose was prepared. C57BL/6J mice carrying the described microbiota composition (LCM, OligoMM12, SPF, see figure legends for specific group numbers) were gavaged with the inoculum (either B. theta WT or B. theta acapsular). Fecal pellets were collected approximately every 4 hr post-inoculation. Fecal pellets were weighted and homogenized in 500 µL of PBS with steel ball by mixing (25 Hz, 2.5 min) in a TissueLyser (QIAGEN). Serial dilutions were plated for quantification on BHI-blood agar plates supplemented with gentamycin 200 µg/L and erythromycin 25 µg/L and cultured in anaerobic conditions. Note that zero colonies grew from the feces of LCM, OligoMM12, and SPF mice on BHIS plates with gentamycin and erythromycin.

For competition experiments, B. theta strains were grown overnight in 8 mL of BHIS with corresponding antibiotics (erythromycin 25 µg/L or tetracycline 2 µg/L). Each culture was spun down at 3000 × g for 20 min and resuspended in 10 mL of PBS and individual O.D. was measured (cell number estimation 1 O.D. = ~4 × 10⁸ cells/mL). Each strain was adjusted to approximately 5 × 10⁶ CFU/100 µL dose per strain in the inoculum mix (one B. theta untagged and six B. theta barcoded strains). GF mice were gavaged with 100 µL of the inoculum. After 48 hr, fecal and cecal contents were collected. Fecal content was homogenized and plated for quantification as described before. Cecal content was resuspended in 1 mL of PBS and homogenized with steel ball by mixing with the same protocol (25 Hz, 2.5 min). Serial dilutions were prepared and plated in BHI-blood agar plates supplemented with gentamycin plus either erythromycin or tetracycline for CFU quantification of each strain. Like the in vitro competition experiment, we isolated DNA from one of the dilutions used for quantification to assess the competition between B. theta barcoded strains. Relative distribution of the tags was obtained by qPCR. For calculating the competition index among strains with different antibiotic resistances, we divided the bacteria density of the untagged B. theta untagged strain by a sixth of the bacteria density of the total B. theta barcoded strains (as all six barcoded strains were present in the culture).

Stationary-phase B. theta cultures were prepared overnight as described before. Each culture was washed with PBS to remove residual antibiotics and adjusted in the inoculum based on its O.D.600nm. Unless otherwise stated, the untagged strain (B. theta untagged) was present at ~5 × 10⁷ CFUs/100 µL dose of the inoculum. For the B. theta barcoded strains, we prepared an initial 1:1:1:1:1:1 mix of all six barcoded strains in 50 mL of PBS at a concentration of 10⁵ CFU/mL of each strain. After mixing by vortex for 1 min, the required amount of B. theta barcoded strains was prepared by serial dilution and spiked into the inoculum (between 30 and 5 × 10⁴ CFUs depending on the experiment). LCM (Oligo) and SPF C57BL/6J mice were gavaged with the 100 µL inoculum. To check composition, the inoculum was serially diluted and plated for quantification of CFUs in BHI-blood agar plates supplemented with gentamycin 200 µg/L plus either erythromycin 25 µg/L or tetracycline 2 µg/L. In addition, three whole doses (100 µL) were directly plated on three BHI-blood agar plates with gentamycin plus erythromycin or tetracycline (as appropriate) to address initial distribution of B. theta barcoded strains in the inoculum by quantitative PCR (qPCR). Unless otherwise stated, 2 days after colonization, mice were euthanized and cecal content was collected in 2 mL Eppendorf tubes and weighted. Cecal content was homogenized as described before. Serial dilutions were prepared and plated in BHI-blood agar plates supplemented with gentamycin plus either erythromycin or tetracycline for CFU quantification. In addition, 100 µL of homogenized content was plated directly in BHI-blood agar plates with gentamycin plus erythromycin to generate biomass of the assessment of the distribution of B. theta barcoded strains by qPCR.

To discard potential increased colonization fitness in the barcoded strains that were present in the cecum content after 48 hr, we isolated single B. theta WT barcoded strains that were present in the cecal content of SPF mice during a colonization experiment. Single colonies were expanded in liquid media, and the presence of a single strain was confirmed by qPCR analysis of the barcodes present. We randomly selected three of the B. theta WT barcoded strains isolated from the cecal content. We prepared an inoculum as described before for the in vivo competition experiments with approximately 5 × 10⁶ CFU/100 µL of each strain in the inoculum mix. We complemented the inoculum with the remaining three B. theta WT barcoded strains coming from the original stock. SPF mice were inoculated by gavage and cecal content was collected 48 hr later. Cecal content was processed as described before for CFU quantification and relative barcode distribution by qPCR.

In accordance with what we described before, B. theta WT strains were grown overnight in BHIS with corresponding antibiotics. As the untagged strain B. theta untagged was used in higher concentrations, we prepared between 50 and 100 mL of liquid culture depending on the number of mice to colonize. Inoculum was prepared as previously described with a concentration of 10⁸–10⁹ CFUs/100 µL dose of untagged B. theta untagged, spiked with approximately 30 CFU of each B. theta barcoded strains. GF mice were gavaged with 100 µL of the inoculum. Mice were maintained on standard chow diet (Kliba Nafag, 3537; autoclaved; per weight: 4.5% fat, 18.5% protein, ~50% carbohydrates, 4.5% fiber) for 4 days. Afterward, mice were housed on fresh IsoCages, and challenges were applied as follows: (1) control group (continuation of standard chow diet); (2) Western-type diet without fiber (BioServ, S3282; 60% kcal fat; irradiated; per weight: 36% fat, 20.5% protein, 35.7% carbohydrates, 0% fiber); or (3) infection with 5 × 10⁷ CFU of attenuated Salmonella Typhimurium (Stm SL1344^ΔSPI-2). Fecal pellets were collected pre-challenge (day 0) and during the following 3 days. On day 3, mice were euthanized and cecal content was collected. Fecal pellets were weighted and homogenized in 500 µL of PBS as described before. Serial dilutions were prepared and plated in BHI-blood agar plates supplemented with corresponding antibiotics for CFU quantification. In addition, 100–300 µL of homogenized content was plated directly for further assessment of the distribution of B. theta barcoded strains by qPCR. Cecal content was processed as previously described.

To assess microbial community composition, fecal pellets from LCM mice and cecum content from SPF mice were obtained and flash frozen. To generate growth estimates of B. theta in an OligoMM12 background, both flash-frozen fecal pellets and cecum content were used. For enzymatic lysis, half a fecal pellet or roughly 30 mg of flash-frozen cecum content per sample were incubated in 100 μL of 1× TE buffer (30 mM Tris-HCl and 1 mM EDTA) supplemented with 30 mg/mL Lysozyme (Sigma-Aldrich), 1.6 U/mL Proteinase K (New England Biolabs), 10 U/mL Mutanolysin (Sigma-Aldrich), and 1 U/μL SUPERase•In RNase Inhibitor (Invitrogen) at room temperature for 10 min. To aid disruption, one 2 mm metal bead was added, and the samples were vortexed every 2 min. Subsequently, the samples were mixed with 550 μL RLT Plus buffer (QIAGEN) complemented with 5.5 μL 2-beta-mercaptoethanol (Sigma-Aldrich) and prefilled tubes with 100 μm Zirconium beads (OPS Diagnostics LLC). The samples were disrupted twice at 30 Hz for 3 min using the mixer mill Retsch MM400 with 5 min incubation at room temperature between each disruption. DNA was extracted from all samples with the DNA/RNA Mini kit (QIAGEN) following the standard protocol and eluting the DNA in 100 μL elution buffer (EB). For the LCM samples, three negative extraction controls and three negative PCR controls were included. For the SPF samples, one water sample was used as negative extraction control and subsequently split into three negative library controls undergoing the same library preparation as all samples. The integrity and quality of the extracted DNA was assessed on a Qubit and Fragment Analyzer, respectively. The DNA was purified by overnight ethanol precipitation at −20°C in 275 μL ice-cold Ethanol (Sigma-Aldrich), 10 μL 3 M sodium acetate (Invitrogen), and 1 μL 20 mg/mL glycogen (Invitrogen) with subsequent centrifugation at 4°C for 30 min and two wash steps in 500 μL ice-cold 75% ethanol with centrifugation at 4°C for 10 min each time. The DNA purity was assessed on a Nanodrop.

16S amplicon libraries were generated from 50 ng input DNA with the Illumina primer set 515F (Parada et al., 2016) and 806R (Apprill et al., 2015), 12 cycles in PCR 1 and 13 cycles in PCR 2. Three positive controls containing 11 ng input DNA of ZymoBIOMICS Microbial Community DNA Standard II (Zymo Research) were used. Illumina Unique Dual Indexing Primers (UDP) were used for library multiplexing. A 12 pM library pool spiked with 20% PhiX was sequenced at the Functional Genomics Center Zurich (FGCZ) using the MiSeq platform and 2 × 300 bp PE-reads with a target fragment size of 450 bp, resulting in approximately 60,000 and 400,000 reads per sample for the LCM and OligoMM12/SPF sequencing runs, respectively. Raw sequencing data from LCM and SPF mice can be accessed on ENA (https://www.ebi.ac.uk/ena/browser/home) under Project ID PRJEB57876. The OligoMM12 was previously published and can be accessed on ENA under the Project ID PRJEB53981 (Hoces et al., 2022).

Genomic DNA was sheared to a target fragment size of 350 bp length with the ultrasonicator Covaris LE220 following a standard protocol (30 μL volume, 220 W peak incident power, 89 s treatment time). Metagenomic libraries were prepared from 10 ng sheared DNA with the NebNext Ultra II DNA Library Prep Kit for Illumina. Sample-specific adaptations included tenfold adapter dilution, no size selection by adding 1 volume (89 μL) of Cytiva Sera-Mag Select beads in the first cleanup and eight PCR cycles in the amplification step. Nebnext Multiplex Oligos for Illumina (Dual Index Primer Set 1) were used for library multiplexing. The final cleanup was done with a left side size selection by adding 0.7 volumes (35 μL) of Cytiva Sera-Mag Select beads. A 1 nM library pool spiked with 3% PhiX was sequenced at the FGCZ using the NextSeq2000 platform and 2 × 150 bp PE-reads with a target fragment size of 500 bp, resulting in approximately 30,000,000 reads per sample.

Sample size was determined based on previous experiments (Maier et al., 2014; Porter et al., 2017; Wotzka et al., 2019) using at least five mice per group where large effect size was expected. All group sizes are described in the figure legends.

Where errors are expected to be log-normal distributed (e.g., CFU density comparisons), all statistical tests were carried out on log-normalized data. One-way ANOVA followed by Tukey’s honest significance test was used for comparison of three or more groups. For model-inferred parameters, we compared mean and standard deviation calculated as described in the previous section. No data points were omitted from statistical analysis or for the estimation of parameters. Statistical analysis was performed with RStudio v1.2 and R v3.6.