Roles of bacterial growth competition systems in colonization of the murine gut

Roles of bacterial growth competition systems in colonization of the murine gut | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Roles of bacterial growth competition systems in colonization of the murine gut Petra Muir, Jonas Kjellin, Evelina Kess, David Low, Sanna Koskiniemi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9649958/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The gut microbiome is essential for human health. Although the gut microbiota is largely stable at the species level in healthy individuals, strain-level variation remains less understood. Many bacterial strains encode toxin delivery systems that may shape competition within the gut. Here, we investigate how contact-dependent growth inhibition (CDI) and colicins influence intestinal colonization by a competitive murine Escherichia coli isolate, R12. We show that R12 can colonize an intact mouse gut microbiota by displacing resident Enterobacteriaceae , but success depends on multiple interacting factors. CDI systems and colicins provide a competitive advantage against resident E. coli , particularly during early colonization, while metabolic flexibility and access to alternative carbon sources support long-term persistence. Colonization outcomes vary between hosts and are shaped by resident microbiota composition, strain-level competition, and the initial invader-to-resident ratio. Overall, successful gut invasion is determined by the combined effects of bacterial antagonistic systems, metabolic capacity, and ecological context. Figures Figure 1 Figure 2 Figure 3 Figure 4 Main text The gastrointestinal tract of mammals is colonized by a large number of microorganisms, commonly referred to as the normal gut microbiota. The composition of the gut microbiota for host extraction and synthesis of nutrients and metabolites, development of the immune system and preventing colonization of pathogens is increasingly recognized 1 . Although the microbiome in healthy individuals remains relatively stable over time 2 , changes on strain level are not well understood. Strain level variation in e.g. E. coli population are important risk factors for colon cancer (colibactin production) as well as urinary tract infections (uropathogenic strains) 3 – 5 . De novo colonization of the gut involves several challenges, including overcoming the colonization resistance provided by the resident microbiota 6 . The mechanisms behind colonization resistance are not yet fully understood, but most likely involve nutrient competition 7 , 8 , 9 and toxin delivery from established bacteria 10 – 17 , 18 , 19 . Bacterial toxins can be delivered either through secretion to the extracellular milieu, e.g. microcins and colicins, or through direct cell-to-cell toxin delivery as with contact-dependent growth inhibition (CDI) or type 6 secretion systems (T6SS), Reviewed in 20,21,22 . Expression of specific immunity proteins protects toxin-expressing cells from inhibiting their own growth (self-recognition) 20 , 23 , 24 . Previous findings have shown that microcins and T6SS, with broad host-ranges, contribute to bacterial colonization 11 , 19 . In contrast, although CDI was first identified in the gut commensal EC93, its role in gut colonization remains poorly understood 25 , 26 . CDI can be considered a short- and narrow-range weapon, as delivery of toxic effector proteins requires physical contact with targeted cells and interaction with specific outer-membrane proteins that determine host-range 27 . Also colicins have narrow host-range, using the same type of receptors as CDI, but as they are secreted to the extra-cellular milieu their range can be considered longer 20 . To recognize the correct target, specific receptor binding domains (RBDs) in the CdiA and Colicin proteins are used for target cell recognition 20 , 27 – 31 . Following engagement of the target cell receptors, the CdiA-CTs and colicin cytotoxic domains, are translocated into target cells 32 where they display a range of activities, including DNase, RNase and pore-forming functions 20 , 28 , 33 – 35 . To improve our understanding of bacterial host colonialization, we studied the highly competitive E.coli isolate R12 from rat in a murine modell, identifying its toxin-delivery systems and their contribution to colonisation. We find that E. coli R12 is able to colonize an intact mouse gut microbiota, but that its success depends strongly on both its toxin delivery systems and the composition of the resident microbiota. Genome analysis shows that R12 encodes two CDI systems and three colicins, which are all functional and contribute to competitive fitness against other E. coli strains, particularly within the Enterobacteriaceae niche. In vivo experiments demonstrate that wild-type R12 outcompetes resident E. coli more effectively than mutants lacking CDI and/or colicins, especially during later stages of colonization. However, colonization success is highly variable between mice and is influenced by the initial invader-to-resident ratio, resident strain competition systems, and the presence of alternative metabolic capabilities. R12’s ability to utilize unique carbon sources and its metabolic flexibility likely support colonization independently of toxin systems, particularly at lower competitive advantages. In contrast, CDI and colicins are crucial for early dominance and successful invasion when competing with established E. coli strains. Overall, colonization is determined by an interplay of bacterial antagonistic systems, metabolic capacity, resident microbiota composition, and initial population ratios. Together, these findings highlight how metabolic fitness and contact-dependent antagonism synergize to drive colonization—offering a framework for designing targeted microbiome interventions and competitive bacterial therapeutics. E. coli R12 can colonize an occupied niche To determine if E. coli R12 can overcome colonization resistance mediated by an intact normal intestinal microbiota, we inoculated female BALB/c mice with 10 7 rifampicin (RIF)-resistant E. coli R12 by oral gavage and measured the prevalence of R12 in the gut by plating fecal material on RIF plates (Fig. 1 A). E. coli R12 levels decreased for the first few days post infection (p.i.) to increase to around 10 6 CFU on day 8 (Fig. 1 B). This suggests that E. coli R12 is able to colonize the mouse gut without prior niche clearance. Previous studies have shown that the lack of Enterobacteriaceae in normal gut microbiota of mice greatly increases colonization efficacy of Salmonella enterica 36 . However, all mice had Enterobacteriaceae in their feces (Fig. 1 C), suggesting that E. coli R12 is a successful colonizer even when the intestinal niche is already occupied. Identification of the E.coli R12 toxin delivery arsenal To investigate if toxin delivery systems could play a role in the ability of R12 to colonize an occupied niche, we performed whole genome sequencing (Pacific Biosciences). The sequences assembled into three contigs: a 4.8 Mb chromosome; a large 81.8 kb plasmid (pR12.82); and a small 8.6 kb plasmid (Fig. 1 D) (accession: GCA_042192135.1). Bioinformatic analysis of the genome using Bagel 4 37 revealed the presence of two chromosomal cdiBAI loci and three plasmid-encoded colicins, colicins B, M and Y (Fig. 1 D). The CdiA-proteins of the two identified CDI systems show high levels of homology (94.6% aa identity) differing only in their C-terminal toxin domains (Fig. S1 ). The RBDs of CdiA-1 and CdiA-2 are identical and highly homologous (99.3% identity) to the RBD of CdiA UPEC536 (Fig. S2 ), which recognizes OmpC/OmpF as receptor on target cells 27 , 29 . Aligning the CdiA-CTs with toxins of known function suggests that cdiA R12−CT1 encodes an AcrB-dependent ionophore toxin as it is 100% identical to that of E. coli EC93 CdiA EC93−CT2 on protein level (Fig. S3 ) 26 . The toxic activity of CdiA R12−CT2 is unknown as we were unable to find significant homology to previously described proteins. The genes encoding colicin B and colicin M are located on an 82 kb plasmid (Fig. 1 D). Colicins B and M are known to exploit siderophore receptors FepA and FhuA for target cell entry 20 . Colicin B is a pore-forming colicin, causing depolarization of the cytoplasmic membrane, whereas colicin M inhibits peptidoglycan synthesis 38 – 40 . R12 also contained the previously characterized 8.6 kb pColY plasmid. This plasmid was first isolated from the murine commensal E. coli MP1(accession: CP139039) 41 , 42 and later also found in NGF-1 (accession: CP016007)) 43 , 44 , and encodes Colicin Y, another pore-forming colicin that recognizes OmpA as receptor 45 , 46 . Bioinformatic analyses show that E. coli R12 is not MP1 (Table S1 ). Taken together, R12 encodes numerous bacterial toxin delivery systems that could be important for its colonization ability. E. coli R12 has two active CDI systems and produces two colicins To determine whether CDI systems and colicins affect bacterial colonization, we constructed strains lacking CDI systems (∆ cdiBAI1 , ∆ cdiBAI2 ), colicins (∆ colBI , ∆ colMI , ∆ colYI ), all colicins (∆colicins), or all competition systems (∆complete). These strains were competed against E. coli R12 or MG1655 targets lacking competition systems and immunity genes on solid media. Wild-type R12 (WT) outcompeted ∆ cdiBAI1 and ∆ cdiBAI2 mutants by ~ 10-fold (Fig. 2 A), while controls (target cells provided with plasmid encoded CdiI immunity (p cdiI ) or when the inhibitors lacked CDI systems (CDI − ) showed no change. R12 ∆colicins outcompeted MG1655 lacking single or double CDI immunity (p cdiI1 or p cdiI2 alone) by ~ 4-logs (Fig. 2 B). This advantage decreased to ~ 1-log against ∆ ompC targets (lacking CDI receptor) or strains expressing both cdiIs (p cdiI1 + 2 )(Fig. 2 B). The small competitive disadvantage of the ∆ ompC strain is likely caused by a known fitness cost associated with deletion of ompC 27 , as Δcomplete showed similar competition against MG1655 Δ ompC . Thus, both CDI systems are active and use OmpC as receptor. To test colicin activity, R12 was competed against targets lacking individual immunity genes. Only ∆ colYI targets were outcompeted ~ 10-fold by WT R12, but not by R12∆colicins; Δ colBI and Δ colMI targets were unaffected (Fig. 2 C). These results suggest that colicin Y, but not colicins B or M, are active under these conditions. As colicins are secreted to the extra-cellular milieu, we also assessed colicin activity in supernatants. MG1655 or immunity-deficient targets were exposed to overnight R12 culture supernatants. No inhibition occurred against R12 targets (Fig. S4A), but WT supernatant fully inhibited MG1655 (Fig. 2 E). Using receptor knockouts (Δ fhuA colicin M, Δ ompA -colicin Y) and strains producing subsets of colicins, we assigned activity to each toxin. The colicin(s) produced and able to enter target is shown at the top of Fig. 2 D. These combinations show that all three colicins are produced and active in supernatant, collectively inhibiting MG1655 growth. There is a discrepancy in colicin-mediated growth inhibition between competition and supernatant assays. One possible explanation is the presence of full-length LPS on E. coli R12, which can block bacteriocins and antimicrobial peptides from accessing the cell surface via steric hindrance or charge-dependent sequestration 47 . We find that MG1655 with full-length LPS 48 is fully resistant to R12 supernatant (Fig. S4A), as observed for R12. During competition, R12 outcompeted MG1655 with full-length LPS 10-fold (Fig. S4B), similar to the effect observed against R12 targets lacking colicin Y immunity (Fig. 2 C). However, full-length LPS does not block CDI, as CDI-expressing R12 outcompetes MG1655 with full-length LPS, whereas colicin-expressing R12 does not (Fig. S4C). Taken together, colicins contribute to fitness, but target LPS structure modulates their effectiveness. Toxin delivery systems contribute to successful host colonization To determine whether colicin and CDI toxin delivery systems contribute to colonization in a intact microbiota, we infected untreated mice with R12 lacking all five competition systems (∆complete) or WT R12 (Fig. 3 A). Both strains showed similar colonization until day 6 p.i. but days 7–9 WT increased 10- to 100-fold, while ∆complete did not (Fig. 3 A). Thus, toxin delivery systems in R12 are critical for intestinal colonization. Microbiota composition changed during infection. Enterobacteriaceae decreased by day 8 in mice infected with both WT and Δcomplete, while the rest of the microbiota remained relatively stable (Figs. 3 B–C, S6, Table S2 ). In Δcomplete-infected mice, Muribaculaceae increased, whereas Prevotellaceae, Rikenellaceae , and Ruminococcaceae decreased over eight days (Fig. S5). Some differences likely reflect initial microbiota variation, as Δcomplete mice had less Muribaculaceae and more Prevotellaceae at day 0 despite co-housing prior to the infection. R12 colonization varied widely between mice for both WT and Δcomplete (Fig. 3 A). Mice with efficient colonization had higher Enterobacteriaceae levels at the end of the experiment (Fig. S6).16S rDNA sequencing showed microbiota differences between mice, but no taxa differed significantly between efficient and inefficient colonization (Table S2 ). However, two Muribaculaceae species (ASV24 and ASV47) differed between these groups for R12 WT (Fig. 3 D, Table S2 ). This analysis was not possible for Δcomplete, as efficient colonization occurred in only one mouse. Thus, R12 does not alter overall microbiota composition, but the initial microbiota outside Enterobacteriaceae influences E. coli colonization. The toxin delivery systems in R12 are only known to target other Enterobacteriaceae 20 , 27 , 49 . Consistent with this, Enterobacteriaceae , all of which were E. coli , decreased during the infection (Fig. 3 C, Table S2 ). To further characterize this population, we analyzed OmpC variable loop sequences from 40 isolates collected before R12 gavage. A single OmpC-type was found in all mice, termed E. coli NF_6988 (NF – normal flora). Genome sequencing identified several competition systems; colicin E7, microcin PDI, and two cdiBAI loci in NF_6988 (Fig. 3 E) ( PRJNA1145732 / SAMN43077747). We next tested competitions between E. coli NF_6988 and R12 (WT or ∆complete) at different starting ratios. At a 10:1 ratio, R12 outcompeted NF_6988 by about 5-logs (Fig. 3 F). In comparison, at 1:10 ratio, NF_6988 outcompeted R12 by 2–3 logs. At a 1:1 ratio NF_6988 outcompeted R12 ∆complete by ~ 3-logs (Fig. 3 F, pink bar). These results indicate that both initial ratio and toxin repertoire determine competitive outcome. We next examined R12:NF_6988 ratios on day 1 of the mice infection. Ratios > 10 occurred once for WT and twice for Δcomplete. This led to efficient colonization for WT, but not Δcomplete (Fig. S7). Most mice had ratios between 1:1 and 1:10, with little colonization (except one Δcomplete case). In two cases, ratios 10:1) favor R12 colonization, other factors influence outcomes at lower ratios. Other studies indicate that carbon nutrition influences bacterial growth in the mouse intestine 7 , 50 – 52 . To compare carbon-utilization by R12 and the microbiota isolate NF_6988, we used Biolog PM1-2 MicroPlates, profiling 190 carbon sources. E. coli MG1655 was included as a reference. We identified 17 carbon metabolized only by one strain (Fig. 3 G). Out of these, R12 utilized 11 not used by NF_6988. Unlike NF_6988, R12 can use galactitol as sole carbon source, a component of mouse chow shown to influence the co-existence of E. coli and Salmonella 51 , 53 . It is possible that R12’s ability to use galactitol, or other unique carbon sources, may enable it to dominate the E. coli niche persist in the gut even in the absence of competitive systems. Colicins and CdiA toxins both contribute to colonization ability The natural microbiota of mice retrieved from Charles River laboratories contained a competive E. coli isolate, requiring a high R12:NF ratio for R12 to dominate. To investigate how toxin delivery system affect colonization without resident competition, we used mice with a controlled microbiota (Fig. 4 A). Given that E. coli R12 primarily impacts the Enterobacteriaceae niche, we pre-treated the mice with streptomycin to deplete Enterobacteriaceae before colonizing with E. coli MG1655 microbiota lacking known competition systems. Mice were then infected with wildtype or mutant R12 strains lacking CDI and/or colicins. By day 2, R12 WT comprised 90–100% of the E. coli population, and by day 6 it had eliminated MG1655 in all mice (Fig. 4 B). In comparison, MG1655 could not displace an established MG1655 population (Fig. S8). Mutants lacking CDI (ΔCDI) showed bimodal colonization where R12 displaced MG1655 in some mice but remained low in others (Fig. 4 B). Similarly, Δcolicins mutants showed impaired, bimodal colonization. The Δcomplete strain showed reduced early colonization but later reached WT levels (Fig. 4 B). These results suggest that competition systems are important early in colonization, while other factors, such as nutrient use, support persistence. Using Biolog data (Fig. 3 H), R12 was found to grow on seven carbon sources not used by MG1655, indicating that metabolic advantages may drive outgrowth independently of toxin delivery. We then tested whether bimodal colonization reflected differences in initial invader:resident ratios. A significant Spearman correlation between day 0 and day 6 ratios was observed for WT (P = 0.002232) and Δcolicins (P = 0.01667) (Fig. S9AB), but not for ΔCDI (P = 0.09618) or Δcomplete (P = 0.171) (Fig. S9CD). This supports that initial ratios influence colonization when competition systems are present. Overall, competition systems, nutrient utilization, and inoculum size shape colonization success, with the initial invader:resident ratio playing a key role in determining outcome. Discussion The microbiota composition is important for our health, but detailed understanding of how bacteria invade occupied niches is lacking. Here we use E. coli as a proxy, to show that narrow-range bacterial weapons such as CDI systems and colicins are important in bacterial colonization of an intact niche, but that their efficacy largely depends on the toxin arsenal and metabolic abilities of the resident microbiota. In addition, we find that competitive E.coli can invade a niche occupied by resident bacteria with weapons of their own, but that this requires high invader:resident ratio at the initial colonization event. At lower ratios, or when the invader lacks toxin delivery systems, colonization efficacy becomes more erratic and susceptible to other factors. Previous work indicates that metabolic capabilities influence gastrointestinal colonization success 7 , 50 , 51 . We find that R12 can utilize several rare carbon sources, some important in the gut and others not metabolized by competing Enterobacteriaceae . Variation in colonization efficiency may therefore reflect differences in non- Enterobacteriaceae microbiota, affecting the availability of these metabolites in the gut. Notably, two Muribaculaceae species (ASV24 and ASV47) differ between mice with efficient versus poor R12 WT colonization. Muribaculaceae metabolize polysaccharides including plant and host glycans, producing monosaccharides that potentially fuel other resident microbiota 54 . However, metabolic capacity varies across species within the family 55 . For example, Muribaculaceae gordoncarteri show high a-arabinase and a-fucosidase activity 56 , 57 , whereas Heminiphilus faecis ferments mannan and L-arabinose 58 . Thus, it is possible that these particular species of Muribaculaceae produce certain metabolites important for R12 colonization. Further work is needed to determine how strain-specific metabolism shapes competition and colonization. On the other hand, Muribaculaceae also produce short-chain fatty acids and regulate the intestinal barrier function and the immune response 59 . The CdiA protein contains multiple filamentous haemagglutinin (FHA) repeats that both induce production of, and are recognized by, secretory IgA (sIgA). 60–62 . sIgA recognition of bacterial surface molecules results in clumping of the cells, prevention of motility and clearance from the gut via intestinal fluid flow 63 , 64 . The resident E.coli NF_6988 encoded similar CdiA proteins as R12. Thus, it is possible that in mice with high levels of these particular Muribaculaceae have a different immune response (e.g. lower levels of CdiA responsive sIgA), resulting in improved colonization efficacy of R12. This is further supported by our finding that R12 ∆complete colonizes mice with MG1655 microbiota more effectively than R12 ∆colicins. If CDI-expressing cells (R12 Δcolicins) are more susceptible to sIgA-mediated clumping and clearance than ΔCDI strains, which lack FHA domains recognized by sIgA, this could explain their reduced colonization efficiency relative to Δcomplete. As the prevalence of specific E. coli strains in the microbiota varies between individuals and over time 65 , our susceptibility to specific E.coli strains might also vary. Strain-level variation in Enterobacteriaceae matters because different E. coli strains cause diseases ranging from diarrhea and urinary tract infections to cancer and inflammatory bowel disease 3 , 66 , 67 . Although little is known about such variation in healthy gut microbiomes, travel studies show frequent colonization by antibiotic-resistant strains in high-AMR regions 68 . Most travelers lose these strains after returning, but some retain them for years 69 . Our data suggest colonization success depends on growth competition and nutrient use of invading and resident strains. Narrow-range antagonistic systems in E. coli R12 target Enterobacteriaceae , offering potential for targeted microbiota modulation, such as removing pathogenic or resistant strains. Overall, this work highlights molecular bacterial interactions in shaping strain-level variation and guiding microbiota interventions. Material and methods Strains and growth conditions The bacterial strains used in this study are listed in table S3. E.coli R12 was isolated from the feces of rats acquired from Charles River laboratories. All strains were grown at 37°C and with shaking at 200 rpm. in M9Gly+CAA minimal media: 1x M9 salts supplemented with 1% glycerol, 0.2% cas-amino acids, 2 mM MgSO 4 and 0.1 mM CaCl 2 , or in Lysogeny broth (LB): 10 g l -1 tryptone, 5 g l -1 yeast extract and 10 g l -1 NaCl. Agar was added at 15 g l -1 for solid media. Media were supplemented with antibiotics when applicable as follows: ampicillin 100 mg l -1 , chloramphenicol 12.5 mg l -1 , kanamycin 50 mg l -1 , streptomycin 100 mg l -1 , rifampicin 100 mg l -1 . Whole-genome sequencing Chromosomal DNA from E. coli R12 and NF_6988 were isolated using the Qiagen Genomic-tip 100/G kit according to the manufacturer. PacBio libraries were produced using the SMRTbell™ Template Prep Kit 1.0 according to manufacturer’s instructions (Pacific Biosciences, CA, USA). Libraries were subjected to exo treatment and PB AMPure bead wash procedures for clean-up before size selection with the BluePippin system (Sage Sciences, MA, USA) with a cut-off value of 6500 bp. The libraries were sequenced on the PacBio RS II instrument using C4 chemistry, P6 polymerase and 240-minute movie time in one SMRTcell™. The Pacbio reads were assembled using HGAP3 from SMRTportal (PacBio, CA, USA) with default settings. For NF_6988, barcoding and library preparation were performed using the Native Barcoding Kit 24 V14, followed by sequencing on an R10.4 flow cell in a MinION Mk1c device (Oxford Nanopore, Oxford, UK). Base calling and adapter trimming were performed with Guppy version 6.3.8+d9e0f64 (Oxford Nanopore) using the super-accuracy configuration (dna_r10.4.1_e8.2_260bps_sup). The resulting reads were then assembled using Flye v. 2.9.2-b1786 70 and inspected using Bandage 71 . Long-read polishing was performed using Medaka v. 1.0.3 (https://github.com/nanoporetech/medaka). Both genomes were then annotated with Prokka 1.14.6 72 before being submitted as complete genomes to NCBI. Colicin production assay Bacterial overnight cultures were centrifuged at 3,000xg for 5 minutes and the culture medium supernatant was passed through a 0.2 µm filter to make spent media. Overnight cultures of test strains were diluted 1:100 in M9 minimal medium, mixed with sterile-filtered spent medium at 1:25 ratio and incubated for 4 h at 37°C before measuring the optical density at 600 nm. Spent media were prepared from E. coli R12 wildtype, ∆colicin Y, and ∆colicins. Test strains were E. coli R12 mutants lacking the colicins and corresponding immunities, or E. coli K12 MG1655 lacking fepA , fhuA , ompA , alone or in combinations. Competition assay Overnight cultures of inhibitor and target cells were mixed at a ratio of 10:1 and 20 µl were spotted onto solid M9Gly+CAA minimal media. The cells were co-cultured for 24 h at 37°C before suspended in 1xPBS and plated onto LB solid media containing appropriate antibiotics to enumerate the number of colony-forming units per milliliter of inhibitors and targets. Competitive indices were calculated as the ratio of inhibitors to target cells at the end of the co-culture divided by the ratio at the start of the co-culture. Animal experiments Forty-eight 7-week old female Balb/c (Charles River laboratories distributed by Scanbur) were housed in individual ventilated cages (Tecniplast boxunsfue) at the Swedish National Veterinary Institute . The mice were housed 8 mice per cage in a controlled environment (23°C ± 2, 75 ACH and 12h light and dark cycle). The mice’s diet was given an autoclaved pellet feed with no less than 18% protein, 5% fat and 5% fibre, tap water and the cages was enriched with autoclaved hay, a house and a tunnel. All personal handling the mice were required to change clothes, shoes and wear overall, gloves and cap before entering the room, also gloves were changed between cages. All the mice were handled with non-aversive tunnel handling since it has been shown to reduce stress and anxiety in laboratory mice 73 . After a seven-day acclamation period the mice were split into 4 mice per cage before the start of the experiment. For all inoculums, a single colony of each bacterial strain was grown in LB overnight. The gavage solution was prepared by diluting the overnight culture 1:10 in 1x Phosphate Buffered Saline (PBS) with bicarbonate solution. For the experiment with undisturbed microbiota, mice were orally gavaged with 100 ml E. coli R12 wildtype or ∆complete (10 7 CFU/mouse). To ensure the bacteria did not affect the mice in any negative way the mice were closely watched after treatment and weighed every day to ensure no significant amount of weight were lost. Fecal samples were taken for CFU counts on days -5, -2, 0-9 as well as DNA extraction and metagenomic sequencing on days 0 and 8. For the pre-colonization experiment, mice were given 5 g l -1 streptomycin in drinking water for 7 days. At day -2 p.i., mice were treated with 20 mg streptomycin per mouse by gavage. At day -1 p.i. streptomycin was removed from the water and mice were orally gavaged with E. coli K12 MG1655 (10 7 colony-forming units (CFU)). The following day, challenger bacteria, E. coli R12 wildtype, ∆CDI, ∆colicins or ∆complete were introduced by oral gavage (10 7 CFU). Fecal samples were taken for CFU counts on days -5, -2, 0-9. To determine the colony counts, each day fecal samples were obtained from each mouse. Fecal samples were weighed and resuspended in PBS using a plunger. The samples were serially diluted, plated on MacConkey agar supplemented with appropriate antibiotics, and CFU were determined by colony counting. Biolog PM metabolic phenotype analysis The phenotypic analysis of E. coli MG1655, E. coli R12 and E. coli NF_6988 were conducted using Biolog Phenotype Microarray (PM) plates, PM1 and PM2, following the recommended protocol (Biolog Inc., Hayward, CA, USA). In summary, the bacteria were inoculated in LB liquid media and incubated for 16 h prior to diluting the sample 1:35 in IF-0 to 42%T (OD 600 ~0.17) and subsequently diluted in IF-0+Dye to 85%T (OD 600 ~0.03). 100 µl per well were loaded on PM1 and P2 metabolic plates and the plates were incubated at 37°C, with orbital shaking, in an Infinite M200 microplate reader (Tecan). Absorbance at 590 nm was measured every 15 minutes for 48 h. Ethics : This study was conducted in accordance with Swedish animal welfare laws. All procedures were done according to LASAs good practice guidelines and handling was done with non-aversive tunnel handling to improve animal welfare 73 . The Uppsala Animal Experiments Ethics review board in Uppsala, Sweden approved all mice protocols undertaken in this study under reference no. 5.8.18-5552/2019. Declarations Data Availability Statement: All raw data for the manuscript are freely available either as supplementary excel files (Tables S2 and S3) or at the NCBI data repository for genome sequences (accession numbers are found in the text). Author Contributions: P.M., D.L., and S.K. conceived the study. P.M., E.K., and S. K. performed experiments. P.M., J.K, E.K., and S.K analyzed data. P.M., D.L., and S.K. wrote the manuscript. Acknowledgements: This study was funded by grants from the Swedish research council, the foundation to prevent antibiotic resistance and Uppsala antibiotics center to S.K. We thank Mia Phillipson and Susan Schlegel for insightful input during the writing of this manuscript. Conflicts of interest: The authors declare no conflicts of interest. References Brestoff, J.R. & Artis, D. Commensal bacteria at the interface of host metabolism and the immune system. Nat Immunol 14, 676–84 (2013). Faith, J.J. et al. The Long-Term Stability of the Human Gut Microbiota. Science 341, 1237439 (2013). Mäklin, T. et al. Geographical variation in the incidence of colorectal cancer and urinary tract cancer is associated with population exposure to colibactin-producing Escherichia coli. The Lancet Microbe 6, 101015 (2025). Díaz-Gay, M. et al. Geographic and age variations in mutational processes in colorectal cancer. Nature 643, 230–240 (2025). Thänert, R. et al. 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The Type VI secretion system: a versatile bacterial weapon. Microbiology (Reading) 165, 503–515 (2019). Cuthbert, B.J., Hayes, C.S. & Goulding, C.W. Functional and Structural Diversity of Bacterial Contact-Dependent Growth Inhibition Effectors. Front Mol Biosci 9, 866854 (2022). Allsopp, L.P. & Bernal, P. Killing in the name of: T6SS structure and effector diversity. Microbiology (Reading) 169(2023). Aoki, S.K. et al. Contact-dependent inhibition of growth in Escherichia coli. Science 309, 1245–8 (2005). Waneskog, M. et al. Escherichia coli EC93 deploys two plasmid-encoded class I contact-dependent growth inhibition systems for antagonistic bacterial interactions. Microb Genom 7(2021). Virtanen, P., Waneskog, M. & Koskiniemi, S. Class II contact-dependent growth inhibition (CDI) systems allow for broad-range cross-species toxin delivery within the Enterobacteriaceae family. Mol Microbiol 111, 1109–1125 (2019). Aoki, S.K. et al. Contact-dependent growth inhibition requires the essential outer membrane protein BamA (YaeT) as the receptor and the inner membrane transport protein AcrB. Mol Microbiol 70, 323–40 (2008). Beck, C.M. et al. CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria. PLoS Pathog 12, e1005925 (2016). Ruhe, Z.C. et al. CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria. mBio 8(2017). Halvorsen, T.M. et al. Lipidation of Class IV CdiA Effector Proteins Promotes Target Cell Recognition during Contact-Dependent Growth Inhibition. mBio 12, e0253021 (2021). Ruhe, Z.C. et al. Programmed Secretion Arrest and Receptor-Triggered Toxin Export during Antibacterial Contact-Dependent Growth Inhibition. Cell 175, 921–933 e14 (2018). Aoki, S.K. et al. A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria. Nature 468, 439–42 (2010). Morse, R.P. et al. Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems. Proc Natl Acad Sci U S A 109, 21480–5 (2012). Beck, C.M. et al. CdiA from Enterobacter cloacae delivers a toxic ribosomal RNase into target bacteria. Structure 22, 707 – 18 (2014). Velazquez, E.M. et al. Endogenous Enterobacteriaceae underlie variation in susceptibility to Salmonella infection. Nat Microbiol 4, 1057–1064 (2019). van Heel, A.J. et al. BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins. Nucleic Acids Research 46, W278-W281 (2018). Schaller, K., Holtje, J.V. & Braun, V. Colicin M is an inhibitor of murein biosynthesis. J Bacteriol 152, 994–1000 (1982). Pressler, U., Braun, V., Wittmann-Liebold, B. & Benz, R. Structural and functional properties of colicin B. J Biol Chem 261, 2654–9 (1986). El Ghachi, M. et al. Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors. J Biol Chem 281, 22761–72 (2006). Pick, K., Stothard, P. & Raivio, T.L. Complete genome sequence of Escherichia coli MP1. Microbiology Resource Announcements 13, e01216-23 (2024). Lasaro, M. et al. Escherichia coli Isolate for Studying Colonization of the Mouse Intestine and Its Application to Two-Component Signaling Knockouts. Journal of Bacteriology 196, 1723–1732 (2014). Kotula, J.W. et al. Programmable bacteria detect and record an environmental signal in the mammalian gut. Proc Natl Acad Sci U S A 111, 4838–43 (2014). Ziesack, M. et al. Escherichia coli NGF-1, a Genetically Tractable, Efficiently Colonizing Murine Gut Isolate. Microbiol Resour Announc 7(2018). Riley, M.A. et al. The newly characterized colicin Y provides evidence of positive selection in pore-former colicin diversification. Microbiology (Reading) 146 (Pt 7), 1671–1677 (2000). Bosák, J., Micenkova, L., Dolezalova, M. & Smajs, D. Colicins U and Y inhibit growth of Escherichia coli strains via recognition of conserved OmpA extracellular loop 1. Int J Med Microbiol 306, 486–494 (2016). Sharp, C. et al. O-Antigen-Dependent Colicin Insensitivity of Uropathogenic Escherichia coli. Journal of Bacteriology 201, 10.1128/jb.00545 – 18 (2019). Browning, D.F. et al. Laboratory adapted Escherichia coli K-12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis. Mol Microbiol 87, 939–50 (2013). Ruhe, Z.C., Wallace, A.B., Low, D.A. & Hayes, C.S. Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species. mBio 4(2013). Chang, D.E. et al. Carbon nutrition of Escherichia coli in the mouse intestine. Proc Natl Acad Sci U S A 101, 7427–32 (2004). Gul, E. et al. Differences in carbon metabolic capacity fuel co-existence and plasmid transfer between Salmonella strains in the mouse gut. Cell Host Microbe 31, 1140–1153 e3 (2023). Fabich, A.J. et al. Comparison of carbon nutrition for pathogenic and commensal Escherichia coli strains in the mouse intestine. Infect Immun 76, 1143–52 (2008). Eberl, C. et al. E. coli enhance colonization resistance against Salmonella Typhimurium by competing for galactitol, a context-dependent limiting carbon source. Cell Host Microbe 29, 1680–1692 e7 (2021). Zhu, Y. et al. Exploration of the Muribaculaceae Family in the Gut Microbiota: Diversity, Metabolism, and Function. Nutrients 16(2024). Smith, B.J., Miller, R.A. & Schmidt, T.M. Muribaculaceae Genomes Assembled from Metagenomes Suggest Genetic Drivers of Differential Response to Acarbose Treatment in Mice. mSphere 6, e0085121 (2021). Miyake, S., Ding, Y., Soh, M. & Seedorf, H. Complete Genome Sequence of Duncaniella muris Strain B8, Isolated from the Feces of C57/BL6 Mice. Microbiol Resour Announc 8(2019). Miyake, S., Ding, Y., Soh, M., Low, A. & Seedorf, H. Cultivation and description of Duncaniella dubosii sp. nov., Duncaniella freteri sp. nov. and emended description of the species Duncaniella muris. Int J Syst Evol Microbiol 70, 3105–3110 (2020). Park, J.K. et al. Heminiphilus faecis gen. nov., sp. nov., a member of the family Muribaculaceae, isolated from mouse faeces and emended description of the genus Muribaculum. Antonie Van Leeuwenhoek 114, 275–286 (2021). Ormerod, K.L. et al. Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals. Microbiome 4, 36 (2016). Leininger, E. et al. Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin. J Infect Dis 175, 1423–31 (1997). Di Tommaso, A. et al. Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses. Infect Immun 59, 3313–5 (1991). Poulain-Godefroy, O., Vendeville, C., Locht, C. & Riveau, G. Bordetella pertussis filamentous hemagglutinin delivered by mucosal routes enhances immunoglobulin levels in serum and mucosal fluids. FEMS Immunol Med Microbiol 54, 129–36 (2008). Moor, K. et al. High-avidity IgA protects the intestine by enchaining growing bacteria. Nature 544, 498–502 (2017). Hoces, D., Arnoldini, M., Diard, M., Loverdo, C. & Slack, E. Growing, evolving and sticking in a flowing environment: understanding IgA interactions with bacteria in the gut. Immunology 159, 52–62 (2020). Han, N. et al. Rapid turnover and short-term blooms of Escherichia coli in the human gut. J Bacteriol 206, e0023923 (2024). Geurtsen, J. et al. Genomics and pathotypes of the many faces of Escherichia coli. FEMS Microbiology Reviews 46, fuac031 (2022). Mirsepasi-Lauridsen Hengameh, C., Vallance Bruce, A., Krogfelt Karen, A. & Petersen Andreas, M. Escherichia coli Pathobionts Associated with Inflammatory Bowel Disease. Clinical Microbiology Reviews 32, 10.1128/cmr.00060 – 18 (2019). Worby, C.J. et al. Gut microbiome perturbation, antibiotic resistance, and Escherichia coli strain dynamics associated with international travel: a metagenomic analysis. The Lancet Microbe 4, e790-e799 (2023). Dallman Timothy, J. et al. Prevalence and Persistence of Antibiotic Resistance Determinants in the Gut of Travelers Returning to the United Kingdom is Associated with Colonization by Pathogenic Escherichia coli. Microbiology Spectrum 11, e05185-22 (2023). Kolmogorov, M. et al. metaFlye: scalable long-read metagenome assembly using repeat graphs. Nat Methods 17, 1103–1110 (2020). Wick, R.R., Schultz, M.B., Zobel, J. & Holt, K.E. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 31, 3350–2 (2015). Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–9 (2014). Gouveia, K. & Hurst, J.L. Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice. Sci Rep 9, 20305 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementalmaterialforR12260417.pdf TableS2.xlsx TableS3.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9649958","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":637791928,"identity":"57105957-0aa6-4de2-8870-15d7f0259ff1","order_by":0,"name":"Petra Muir","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"","lastName":"Muir","suffix":""},{"id":637791929,"identity":"9fe44f52-83a3-45a8-b849-29b198e3b737","order_by":1,"name":"Jonas Kjellin","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Jonas","middleName":"","lastName":"Kjellin","suffix":""},{"id":637791930,"identity":"2b857148-a88f-432b-9ad9-8b5647152cae","order_by":2,"name":"Evelina Kess","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Evelina","middleName":"","lastName":"Kess","suffix":""},{"id":637791931,"identity":"7722f48c-065a-4ae0-904d-42f19fc2b14d","order_by":3,"name":"David Low","email":"","orcid":"","institution":"University of California Santa","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Low","suffix":""},{"id":637791932,"identity":"de45dd11-1951-49fb-8ec8-891f6d676503","order_by":4,"name":"Sanna Koskiniemi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYPCCBAYGHsYGMMXPTLIWyWbitUCZBgcIqJVvP3vswQ+GNDn5nsNtDx5UbJMxPs78dANj22GgVANWLQZn8tINexhyjA3ONrYbJJy5zWN2mM3sBkiLwRns9hkw5JhJ8DBUJG7gZ2yTSGwDaeFhg2iRSMDusP43ZpJ/gFrm94O0/LvNY9wM1SI//wF2z9zIMZPmYchJbDjbCNTScJvHgBmqheEGdh0GN96YScsYpBkbnDnYJpFw7DaPBMgvCefSeQzO4HJYjpnkm4pkYIilP5P8UXPbnr//8LMbH8qs5eTbsXsfFghoIAERT6NgFIyCUTAKyAAAfhNaL3JyYtcAAAAASUVORK5CYII=","orcid":"","institution":"Uppsala University","correspondingAuthor":true,"prefix":"","firstName":"Sanna","middleName":"","lastName":"Koskiniemi","suffix":""}],"badges":[],"createdAt":"2026-05-08 06:53:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9649958/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9649958/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109205956,"identity":"05503d66-c688-4b5b-8fef-8c4299669226","added_by":"auto","created_at":"2026-05-13 15:09:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R12 colonization of mice.\u003c/strong\u003e (A) Schematic overview of mouse experiment with intact gut microbiota. (B) Untreated BALB/c mice were inoculated orally with 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eE. coli\u003c/em\u003e R12 and the number of CFU were determined by platin fecal material on MacConkey agar plates with or without Rifampicin. The data is plotted as CFU/g feces relative to day 1. Each dot represents one mouse (n = 8). P values were obtained using Mann-Whitney U test comparing the different days to day 1 p.i. *, p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (C) \u003cem\u003eEnterobacteriaceae\u003c/em\u003e before the infection measured as cfu/ g feces on MacConkey agar plates . (D)\u003cstrong\u003e \u003c/strong\u003eOverview of the \u003cem\u003eE. coli\u003c/em\u003e R12 genome\u003cem\u003e.\u003c/em\u003e Genes encoding CDI systems (\u003cem\u003ecdiBAI\u003c/em\u003e), colicins (\u003cem\u003ecolBI\u003c/em\u003e, \u003cem\u003ecolMI\u003c/em\u003e, \u003cem\u003ecolYI\u003c/em\u003e), transfer proteins (\u003cem\u003etra\u003c/em\u003e), P pili (\u003cem\u003epap\u003c/em\u003e) as well as a vacuolating toxin are indicated.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/415c6256e08398fef10e9491.png"},{"id":109205103,"identity":"5d611741-deec-478f-8d85-696dce0a795c","added_by":"auto","created_at":"2026-05-13 15:03:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColicin and CDI activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eR12.\u003c/strong\u003e (A, B) To check for CDI activity, wild-type \u003cem\u003eE. coli\u003c/em\u003e R12 and deletion mutants were co-cultured for 24 h on M9Gly+casAA solid media with (A) R12 lacking \u003cem\u003ecdiBAI1\u003c/em\u003e or \u003cem\u003ecdiBAI2\u003c/em\u003e or (B) MG1655 lacking \u003cem\u003eompC\u003c/em\u003e or complemented with CDI immunities. (C) Indicated R12 inhibitors were cocultured with R12 targets lacking \u003cem\u003ecolYI\u003c/em\u003e, \u003cem\u003ecolBI\u003c/em\u003eor \u003cem\u003ecolMI\u003c/em\u003e at a 10:1 ratio for 24 h on solid M9Gly+CAA minimal media to check for colicin activity. (D) MG1655 lacking the indicated colicin receptor were incubated with culture supernatant from R12 wt, ∆\u003cem\u003ecolB\u003c/em\u003e, ∆\u003cem\u003ecolY\u003c/em\u003e or ∆colicins for 4 h before measuring optical density. Data are the averages ± SEM of at least three independent experiments. For statistical analyses, two-way ANOVA with Tukey’s correction for multiple comparison (A, C), unpaired t-test (B, E) In D, significance was determined towards the Dcolicin supernatant for each target. ns, not significant, *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, **** ≤ 0.0001, ***** ≤ 0.00001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/a15da1ca76214a294dd9f8f2.png"},{"id":109158031,"identity":"bcf3b87a-aad3-4b41-94a7-e2f149e64f4c","added_by":"auto","created_at":"2026-05-13 07:04:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":305285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZ\u003c/strong\u003e in bold and carbon sources utilized by R12 but not MG1655 are highlighted in red.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/27eb4d76f49ada6cb072adf6.png"},{"id":109158034,"identity":"c1811570-1d17-4a6a-903d-72b0afc2e29b","added_by":"auto","created_at":"2026-05-13 07:04:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColonization is dependent on CDI and colicins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic overview of mouse experiment. Antibiotic-treated BALB/c mice were orally inoculated with 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eE. coli\u003c/em\u003e K12 MG1655 and subsequently challenged with 10\u003csup\u003e7\u003c/sup\u003e of either wildtype or mutant \u003cem\u003eE. coli\u003c/em\u003e R12. Each day fecal samples were collected and CFU counts were determined. (B) Mice pre-colonized with MG1655 were challenged with wildtype, or mutants lacking both CDI systems (∆CDI), all three colicins (∆colicins) or CDI systems and colicins (∆complete), and CFU counts of MG1655 and R12 were determined from fecal samples. Each dot represents one mouse. P values were obtained using Mann-Whitney U test comparing the mutant strains to WT. *, p ≤ 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/32cc9cc114ec3b01492bd9c5.png"},{"id":109249643,"identity":"6521fe58-e8a1-4bc0-b48a-3267cf76e903","added_by":"auto","created_at":"2026-05-14 08:58:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1009158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/1bf3811d-dd54-4962-9463-9b6f0c919131.pdf"},{"id":109158029,"identity":"14a058d5-080e-40dc-8b4b-b0658071f385","added_by":"auto","created_at":"2026-05-13 07:04:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1387353,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalmaterialforR12260417.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/7901a04a13046f63d91c1513.pdf"},{"id":109222361,"identity":"c946f7f7-04d5-4508-ae08-0fd4cf76f306","added_by":"auto","created_at":"2026-05-13 21:08:28","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1185909,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/b9561aef2d821166d4058e13.xlsx"},{"id":109205312,"identity":"52f8cff2-a3e9-4541-8d3d-b707cce8d1bd","added_by":"auto","created_at":"2026-05-13 15:04:06","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":310255,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9649958/v1/582adc8e875ab2bb6498995c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Roles of bacterial growth competition systems in colonization of the murine gut","fulltext":[{"header":"Main text","content":"\u003cp\u003eThe gastrointestinal tract of mammals is colonized by a large number of microorganisms, commonly referred to as the normal gut microbiota. The composition of the gut microbiota for host extraction and synthesis of nutrients and metabolites, development of the immune system and preventing colonization of pathogens is increasingly recognized \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although the microbiome in healthy individuals remains relatively stable over time \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, changes on strain level are not well understood. Strain level variation in e.g. \u003cem\u003eE. coli\u003c/em\u003e population are important risk factors for colon cancer (colibactin production) as well as urinary tract infections (uropathogenic strains) \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e colonization of the gut involves several challenges, including overcoming the colonization resistance provided by the resident microbiota \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The mechanisms behind colonization resistance are not yet fully understood, but most likely involve nutrient competition \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and toxin delivery from established bacteria \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Bacterial toxins can be delivered either through secretion to the extracellular milieu, e.g. microcins and colicins, or through direct cell-to-cell toxin delivery as with contact-dependent growth inhibition (CDI) or type 6 secretion systems (T6SS), Reviewed in \u003csup\u003e20,21,22\u003c/sup\u003e. Expression of specific immunity proteins protects toxin-expressing cells from inhibiting their own growth (self-recognition)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Previous findings have shown that microcins and T6SS, with broad host-ranges, contribute to bacterial colonization \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In contrast, although CDI was first identified in the gut commensal EC93, its role in gut colonization remains poorly understood \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCDI can be considered a short- and narrow-range weapon, as delivery of toxic effector proteins requires physical contact with targeted cells and interaction with specific outer-membrane proteins that determine host-range\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Also colicins have narrow host-range, using the same type of receptors as CDI, but as they are secreted to the extra-cellular milieu their range can be considered longer \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To recognize the correct target, specific receptor binding domains (RBDs) in the CdiA and Colicin proteins are used for target cell recognition \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Following engagement of the target cell receptors, the CdiA-CTs and colicin cytotoxic domains, are translocated into target cells \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e where they display a range of activities, including DNase, RNase and pore-forming functions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo improve our understanding of bacterial host colonialization, we studied the highly competitive \u003cem\u003eE.coli\u003c/em\u003e isolate R12 from rat in a murine modell, identifying its toxin-delivery systems and their contribution to colonisation. We find that \u003cem\u003eE. coli\u003c/em\u003e R12 is able to colonize an intact mouse gut microbiota, but that its success depends strongly on both its toxin delivery systems and the composition of the resident microbiota. Genome analysis shows that R12 encodes two CDI systems and three colicins, which are all functional and contribute to competitive fitness against other \u003cem\u003eE. coli\u003c/em\u003e strains, particularly within the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e niche. \u003cem\u003eIn vivo\u003c/em\u003e experiments demonstrate that wild-type R12 outcompetes resident \u003cem\u003eE. coli\u003c/em\u003e more effectively than mutants lacking CDI and/or colicins, especially during later stages of colonization. However, colonization success is highly variable between mice and is influenced by the initial invader-to-resident ratio, resident strain competition systems, and the presence of alternative metabolic capabilities. R12\u0026rsquo;s ability to utilize unique carbon sources and its metabolic flexibility likely support colonization independently of toxin systems, particularly at lower competitive advantages. In contrast, CDI and colicins are crucial for early dominance and successful invasion when competing with established \u003cem\u003eE. coli\u003c/em\u003e strains. Overall, colonization is determined by an interplay of bacterial antagonistic systems, metabolic capacity, resident microbiota composition, and initial population ratios. Together, these findings highlight how metabolic fitness and contact-dependent antagonism synergize to drive colonization\u0026mdash;offering a framework for designing targeted microbiome interventions and competitive bacterial therapeutics.\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eR12 can colonize an occupied niche\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine if \u003cem\u003eE. coli\u003c/em\u003e R12 can overcome colonization resistance mediated by an intact normal intestinal microbiota, we inoculated female BALB/c mice with 10\u003csup\u003e7\u003c/sup\u003e rifampicin (RIF)-resistant \u003cem\u003eE. coli\u003c/em\u003e R12 by oral gavage and measured the prevalence of R12 in the gut by plating fecal material on RIF plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). \u003cem\u003eE. coli\u003c/em\u003e R12 levels decreased for the first few days post infection (p.i.) to increase to around 10\u003csup\u003e6\u003c/sup\u003e CFU on day 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This suggests that \u003cem\u003eE. coli\u003c/em\u003e R12 is able to colonize the mouse gut without prior niche clearance. Previous studies have shown that the lack of \u003cem\u003eEnterobacteriaceae\u003c/em\u003e in normal gut microbiota of mice greatly increases colonization efficacy of \u003cem\u003eSalmonella enterica\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, all mice had \u003cem\u003eEnterobacteriaceae\u003c/em\u003e in their feces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting that \u003cem\u003eE. coli\u003c/em\u003e R12 is a successful colonizer even when the intestinal niche is already occupied.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003eE.coli\u003c/b\u003e \u003cb\u003eR12 toxin delivery arsenal\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate if toxin delivery systems could play a role in the ability of R12 to colonize an occupied niche, we performed whole genome sequencing (Pacific Biosciences). The sequences assembled into three contigs: a 4.8 Mb chromosome; a large 81.8 kb plasmid (pR12.82); and a small 8.6 kb plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) (accession: GCA_042192135.1). Bioinformatic analysis of the genome using Bagel 4\u003csup\u003e37\u003c/sup\u003e revealed the presence of two chromosomal \u003cem\u003ecdiBAI\u003c/em\u003e loci and three plasmid-encoded colicins, colicins B, M and Y (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe CdiA-proteins of the two identified CDI systems show high levels of homology (94.6% aa identity) differing only in their C-terminal toxin domains (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The RBDs of CdiA-1 and CdiA-2 are identical and highly homologous (99.3% identity) to the RBD of CdiA\u003csup\u003eUPEC536\u003c/sup\u003e (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), which recognizes OmpC/OmpF as receptor on target cells \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Aligning the CdiA-CTs with toxins of known function suggests that \u003cem\u003ecdiA\u003c/em\u003e\u003csup\u003eR12\u0026minus;CT1\u003c/sup\u003e encodes an AcrB-dependent ionophore toxin as it is 100% identical to that of \u003cem\u003eE. coli\u003c/em\u003e EC93 CdiA\u003csup\u003eEC93\u0026minus;CT2\u003c/sup\u003e on protein level (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The toxic activity of CdiA\u003csup\u003eR12\u0026minus;CT2\u003c/sup\u003e is unknown as we were unable to find significant homology to previously described proteins.\u003c/p\u003e \u003cp\u003eThe genes encoding colicin B and colicin M are located on an 82 kb plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Colicins B and M are known to exploit siderophore receptors FepA and FhuA for target cell entry \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Colicin B is a pore-forming colicin, causing depolarization of the cytoplasmic membrane, whereas colicin M inhibits peptidoglycan synthesis \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. R12 also contained the previously characterized 8.6 kb pColY plasmid. This plasmid was first isolated from the murine commensal \u003cem\u003eE. coli\u003c/em\u003e MP1(accession: CP139039) \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and later also found in NGF-1 (accession: CP016007))\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and encodes Colicin Y, another pore-forming colicin that recognizes OmpA as receptor \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Bioinformatic analyses show that \u003cem\u003eE. coli\u003c/em\u003e R12 is not MP1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Taken together, R12 encodes numerous bacterial toxin delivery systems that could be important for its colonization ability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eR12 has two active CDI systems and produces two colicins\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether CDI systems and colicins affect bacterial colonization, we constructed strains lacking CDI systems (∆\u003cem\u003ecdiBAI1\u003c/em\u003e, ∆\u003cem\u003ecdiBAI2\u003c/em\u003e), colicins (∆\u003cem\u003ecolBI\u003c/em\u003e, ∆\u003cem\u003ecolMI\u003c/em\u003e, ∆\u003cem\u003ecolYI\u003c/em\u003e), all colicins (∆colicins), or all competition systems (∆complete). These strains were competed against \u003cem\u003eE. coli\u003c/em\u003e R12 or MG1655 targets lacking competition systems and immunity genes on solid media.\u003c/p\u003e \u003cp\u003eWild-type R12 (WT) outcompeted ∆\u003cem\u003ecdiBAI1\u003c/em\u003e and ∆\u003cem\u003ecdiBAI2\u003c/em\u003e mutants by ~\u0026thinsp;10-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while controls (target cells provided with plasmid encoded CdiI immunity (p\u003cem\u003ecdiI\u003c/em\u003e) or when the inhibitors lacked CDI systems (CDI\u003csup\u003e\u0026minus;\u003c/sup\u003e) showed no change.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eR12 ∆colicins outcompeted MG1655 lacking single or double CDI immunity (p\u003cem\u003ecdiI1\u003c/em\u003e or p\u003cem\u003ecdiI2\u003c/em\u003e alone) by ~\u0026thinsp;4-logs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This advantage decreased to ~\u0026thinsp;1-log against ∆\u003cem\u003eompC\u003c/em\u003e targets (lacking CDI receptor) or strains expressing both \u003cem\u003ecdiIs\u003c/em\u003e (p\u003cem\u003ecdiI1\u0026thinsp;+\u0026thinsp;2\u003c/em\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The small competitive disadvantage of the ∆\u003cem\u003eompC\u003c/em\u003e strain is likely caused by a known fitness cost associated with deletion of \u003cem\u003eompC\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, as Δcomplete showed similar competition against MG1655 Δ\u003cem\u003eompC\u003c/em\u003e. Thus, both CDI systems are active and use OmpC as receptor.\u003c/p\u003e \u003cp\u003eTo test colicin activity, R12 was competed against targets lacking individual immunity genes. Only ∆\u003cem\u003ecolYI\u003c/em\u003e targets were outcompeted\u0026thinsp;~\u0026thinsp;10-fold by WT R12, but not by R12∆colicins; Δ\u003cem\u003ecolBI\u003c/em\u003e and Δ\u003cem\u003ecolMI\u003c/em\u003e targets were unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results suggest that colicin Y, but not colicins B or M, are active under these conditions.\u003c/p\u003e \u003cp\u003eAs colicins are secreted to the extra-cellular milieu, we also assessed colicin activity in supernatants. MG1655 or immunity-deficient targets were exposed to overnight R12 culture supernatants. No inhibition occurred against R12 targets (Fig. S4A), but WT supernatant fully inhibited MG1655 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Using receptor knockouts (Δ\u003cem\u003efhuA\u003c/em\u003e colicin M, Δ\u003cem\u003eompA\u003c/em\u003e -colicin Y) and strains producing subsets of colicins, we assigned activity to each toxin. The colicin(s) produced and able to enter target is shown at the top of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. These combinations show that all three colicins are produced and active in supernatant, collectively inhibiting MG1655 growth.\u003c/p\u003e \u003cp\u003eThere is a discrepancy in colicin-mediated growth inhibition between competition and supernatant assays. One possible explanation is the presence of full-length LPS on \u003cem\u003eE. coli\u003c/em\u003e R12, which can block bacteriocins and antimicrobial peptides from accessing the cell surface via steric hindrance or charge-dependent sequestration \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. We find that MG1655 with full-length LPS \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e is fully resistant to R12 supernatant (Fig. S4A), as observed for R12. During competition, R12 outcompeted MG1655 with full-length LPS 10-fold (Fig. S4B), similar to the effect observed against R12 targets lacking colicin Y immunity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, full-length LPS does not block CDI, as CDI-expressing R12 outcompetes MG1655 with full-length LPS, whereas colicin-expressing R12 does not (Fig. S4C). Taken together, colicins contribute to fitness, but target LPS structure modulates their effectiveness.\u003c/p\u003e\n\u003ch3\u003eToxin delivery systems contribute to successful host colonization\u003c/h3\u003e\n\u003cp\u003eTo determine whether colicin and CDI toxin delivery systems contribute to colonization in a intact microbiota, we infected untreated mice with R12 lacking all five competition systems (∆complete) or WT R12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Both strains showed similar colonization until day 6 p.i. but days 7\u0026ndash;9 WT increased 10- to 100-fold, while ∆complete did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Thus, toxin delivery systems in R12 are critical for intestinal colonization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicrobiota composition changed during infection. \u003cem\u003eEnterobacteriaceae\u003c/em\u003e decreased by day 8 in mice infected with both WT and Δcomplete, while the rest of the microbiota remained relatively stable (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;C, S6, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In Δcomplete-infected mice, \u003cem\u003eMuribaculaceae\u003c/em\u003e increased, whereas \u003cem\u003ePrevotellaceae, Rikenellaceae\u003c/em\u003e, and \u003cem\u003eRuminococcaceae\u003c/em\u003e decreased over eight days (Fig. S5). Some differences likely reflect initial microbiota variation, as Δcomplete mice had less \u003cem\u003eMuribaculaceae\u003c/em\u003e and more \u003cem\u003ePrevotellaceae\u003c/em\u003e at day 0 despite co-housing prior to the infection.\u003c/p\u003e \u003cp\u003eR12 colonization varied widely between mice for both WT and Δcomplete (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Mice with efficient colonization had higher \u003cem\u003eEnterobacteriaceae\u003c/em\u003e levels at the end of the experiment (Fig. S6).16S rDNA sequencing showed microbiota differences between mice, but no taxa differed significantly between efficient and inefficient colonization (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, two \u003cem\u003eMuribaculaceae\u003c/em\u003e species (ASV24 and ASV47) differed between these groups for R12 WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This analysis was not possible for Δcomplete, as efficient colonization occurred in only one mouse. Thus, R12 does not alter overall microbiota composition, but the initial microbiota outside \u003cem\u003eEnterobacteriaceae\u003c/em\u003e influences \u003cem\u003eE. coli\u003c/em\u003e colonization.\u003c/p\u003e \u003cp\u003eThe toxin delivery systems in R12 are only known to target other \u003cem\u003eEnterobacteriaceae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Consistent with this, \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, all of which were \u003cem\u003eE. coli\u003c/em\u003e, decreased during the infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). To further characterize this population, we analyzed OmpC variable loop sequences from 40 isolates collected before R12 gavage. A single OmpC-type was found in all mice, termed \u003cem\u003eE. coli\u003c/em\u003e NF_6988 (NF \u0026ndash; normal flora). Genome sequencing identified several competition systems; colicin E7, microcin PDI, and two \u003cem\u003ecdiBAI\u003c/em\u003e loci in NF_6988 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) ( PRJNA1145732 / SAMN43077747). We next tested competitions between \u003cem\u003eE. coli\u003c/em\u003e NF_6988 and R12 (WT or ∆complete) at different starting ratios. At a 10:1 ratio, R12 outcompeted NF_6988 by about 5-logs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In comparison, at 1:10 ratio, NF_6988 outcompeted R12 by 2\u0026ndash;3 logs. At a 1:1 ratio NF_6988 outcompeted R12 ∆complete by ~\u0026thinsp;3-logs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, pink bar). These results indicate that both initial ratio and toxin repertoire determine competitive outcome.\u003c/p\u003e \u003cp\u003eWe next examined R12:NF_6988 ratios on day 1 of the mice infection. Ratios\u0026thinsp;\u0026gt;\u0026thinsp;10 occurred once for WT and twice for Δcomplete. This led to efficient colonization for WT, but not Δcomplete (Fig. S7). Most mice had ratios between 1:1 and 1:10, with little colonization (except one Δcomplete case). In two cases, ratios\u0026thinsp;\u0026lt;\u0026thinsp;1:10 still led to efficient colonization. However, no significant Spearman correlation was found between initial ratio and colonization success (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Thus, while high initial ratios (\u0026gt;\u0026thinsp;10:1) favor R12 colonization, other factors influence outcomes at lower ratios.\u003c/p\u003e \u003cp\u003eOther studies indicate that carbon nutrition influences bacterial growth in the mouse intestine \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. To compare carbon-utilization by R12 and the microbiota isolate NF_6988, we used Biolog PM1-2 MicroPlates, profiling 190 carbon sources. \u003cem\u003eE. coli\u003c/em\u003e MG1655 was included as a reference. We identified 17 carbon metabolized only by one strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Out of these, R12 utilized 11 not used by NF_6988. Unlike NF_6988, R12 can use galactitol as sole carbon source, a component of mouse chow shown to influence the co-existence of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. It is possible that R12\u0026rsquo;s ability to use galactitol, or other unique carbon sources, may enable it to dominate the \u003cem\u003eE. coli\u003c/em\u003e niche persist in the gut even in the absence of competitive systems.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eColicins and CdiA toxins both contribute to colonization ability\u003c/h2\u003e \u003cp\u003eThe natural microbiota of mice retrieved from Charles River laboratories contained a competive \u003cem\u003eE. coli\u003c/em\u003e isolate, requiring a high R12:NF ratio for R12 to dominate. To investigate how toxin delivery system affect colonization without resident competition, we used mice with a controlled microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Given that \u003cem\u003eE. coli\u003c/em\u003e R12 primarily impacts the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e niche, we pre-treated the mice with streptomycin to deplete \u003cem\u003eEnterobacteriaceae\u003c/em\u003e before colonizing with \u003cem\u003eE. coli\u003c/em\u003e MG1655 microbiota lacking known competition systems. Mice were then infected with wildtype or mutant R12 strains lacking CDI and/or colicins. By day 2, R12 WT comprised 90\u0026ndash;100% of the \u003cem\u003eE. coli\u003c/em\u003e population, and by day 6 it had eliminated MG1655 in all mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In comparison, MG1655 could not displace an established MG1655 population (Fig. S8). Mutants lacking CDI (ΔCDI) showed bimodal colonization where R12 displaced MG1655 in some mice but remained low in others (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similarly, Δcolicins mutants showed impaired, bimodal colonization. The Δcomplete strain showed reduced early colonization but later reached WT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggest that competition systems are important early in colonization, while other factors, such as nutrient use, support persistence. Using Biolog data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), R12 was found to grow on seven carbon sources not used by MG1655, indicating that metabolic advantages may drive outgrowth independently of toxin delivery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then tested whether bimodal colonization reflected differences in initial invader:resident ratios. A significant Spearman correlation between day 0 and day 6 ratios was observed for WT (P\u0026thinsp;=\u0026thinsp;0.002232) and Δcolicins (P\u0026thinsp;=\u0026thinsp;0.01667) (Fig. S9AB), but not for ΔCDI (P\u0026thinsp;=\u0026thinsp;0.09618) or Δcomplete (P\u0026thinsp;=\u0026thinsp;0.171) (Fig. S9CD). This supports that initial ratios influence colonization when competition systems are present. Overall, competition systems, nutrient utilization, and inoculum size shape colonization success, with the initial invader:resident ratio playing a key role in determining outcome.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe microbiota composition is important for our health, but detailed understanding of how bacteria invade occupied niches is lacking. Here we use \u003cem\u003eE. coli\u003c/em\u003e as a proxy, to show that narrow-range bacterial weapons such as CDI systems and colicins are important in bacterial colonization of an intact niche, but that their efficacy largely depends on the toxin arsenal and metabolic abilities of the resident microbiota. In addition, we find that competitive \u003cem\u003eE.coli\u003c/em\u003e can invade a niche occupied by resident bacteria with weapons of their own, but that this requires high invader:resident ratio at the initial colonization event. At lower ratios, or when the invader lacks toxin delivery systems, colonization efficacy becomes more erratic and susceptible to other factors.\u003c/p\u003e \u003cp\u003ePrevious work indicates that metabolic capabilities influence gastrointestinal colonization success \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. We find that R12 can utilize several rare carbon sources, some important in the gut and others not metabolized by competing \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. Variation in colonization efficiency may therefore reflect differences in non- \u003cem\u003eEnterobacteriaceae\u003c/em\u003e microbiota, affecting the availability of these metabolites in the gut. Notably, two \u003cem\u003eMuribaculaceae\u003c/em\u003e species (ASV24 and ASV47) differ between mice with efficient versus poor R12 WT colonization. \u003cem\u003eMuribaculaceae\u003c/em\u003e metabolize polysaccharides including plant and host glycans, producing monosaccharides that potentially fuel other resident microbiota \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, metabolic capacity varies across species within the family \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. For example, \u003cem\u003eMuribaculaceae gordoncarteri\u003c/em\u003e show high a-arabinase and a-fucosidase activity \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, whereas \u003cem\u003eHeminiphilus faecis\u003c/em\u003e ferments mannan and L-arabinose \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Thus, it is possible that these particular species of \u003cem\u003eMuribaculaceae\u003c/em\u003e produce certain metabolites important for R12 colonization. Further work is needed to determine how strain-specific metabolism shapes competition and colonization.\u003c/p\u003e \u003cp\u003eOn the other hand, \u003cem\u003eMuribaculaceae\u003c/em\u003e also produce short-chain fatty acids and regulate the intestinal barrier function and the immune response \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The CdiA protein contains multiple filamentous haemagglutinin (FHA) repeats that both induce production of, and are recognized by, secretory IgA (sIgA). \u003csup\u003e60\u0026ndash;62\u003c/sup\u003e. sIgA recognition of bacterial surface molecules results in clumping of the cells, prevention of motility and clearance from the gut via intestinal fluid flow \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The resident \u003cem\u003eE.coli\u003c/em\u003e NF_6988 encoded similar CdiA proteins as R12. Thus, it is possible that in mice with high levels of these particular \u003cem\u003eMuribaculaceae\u003c/em\u003e have a different immune response (e.g. lower levels of CdiA responsive sIgA), resulting in improved colonization efficacy of R12. This is further supported by our finding that R12 ∆complete colonizes mice with MG1655 microbiota more effectively than R12 ∆colicins. If CDI-expressing cells (R12 Δcolicins) are more susceptible to sIgA-mediated clumping and clearance than ΔCDI strains, which lack FHA domains recognized by sIgA, this could explain their reduced colonization efficiency relative to Δcomplete. As the prevalence of specific \u003cem\u003eE. coli\u003c/em\u003e strains in the microbiota varies between individuals and over time \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, our susceptibility to specific \u003cem\u003eE.coli\u003c/em\u003e strains might also vary.\u003c/p\u003e \u003cp\u003eStrain-level variation in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e matters because different \u003cem\u003eE. coli\u003c/em\u003e strains cause diseases ranging from diarrhea and urinary tract infections to cancer and inflammatory bowel disease \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Although little is known about such variation in healthy gut microbiomes, travel studies show frequent colonization by antibiotic-resistant strains in high-AMR regions \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Most travelers lose these strains after returning, but some retain them for years \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Our data suggest colonization success depends on growth competition and nutrient use of invading and resident strains. Narrow-range antagonistic systems in \u003cem\u003eE. coli\u003c/em\u003e R12 target \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, offering potential for targeted microbiota modulation, such as removing pathogenic or resistant strains. Overall, this work highlights molecular bacterial interactions in shaping strain-level variation and guiding microbiota interventions.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial strains used in this study are listed in table S3. \u003cem\u003eE.coli\u003c/em\u003e R12 was isolated from the feces of rats acquired from Charles River laboratories. All strains were grown at 37°C and with shaking at 200 rpm. in M9Gly+CAA minimal media: 1x M9 salts supplemented with 1% glycerol, 0.2% cas-amino acids, 2 mM MgSO\u003csub\u003e4\u003c/sub\u003e and 0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, or in Lysogeny broth (LB): 10 g l\u003csup\u003e-1\u003c/sup\u003e tryptone, 5 g l\u003csup\u003e-1\u003c/sup\u003e yeast extract and 10 g l\u003csup\u003e-1\u003c/sup\u003e NaCl. Agar was added at 15 g l\u003csup\u003e-1\u003c/sup\u003e for solid media. Media were supplemented with antibiotics when applicable as follows: ampicillin 100 mg l\u003csup\u003e-1\u003c/sup\u003e, chloramphenicol 12.5 mg l\u003csup\u003e-1\u003c/sup\u003e, kanamycin 50 mg l\u003csup\u003e-1\u003c/sup\u003e, streptomycin 100 mg l\u003csup\u003e-1\u003c/sup\u003e, rifampicin 100 mg l\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromosomal DNA from \u003cem\u003eE. coli\u003c/em\u003e R12 and NF_6988 were isolated using the Qiagen Genomic-tip 100/G kit according to the manufacturer. PacBio libraries were produced using the SMRTbell™ Template Prep Kit 1.0 according to manufacturer’s instructions (Pacific Biosciences, CA, USA).\u0026nbsp;Libraries were subjected to exo treatment and PB AMPure bead wash procedures for clean-up\u0026nbsp;before size selection with the BluePippin system (Sage Sciences, MA, USA) with a cut-off value of 6500 bp. The libraries were sequenced on the PacBio RS II instrument using C4 chemistry, P6 polymerase and 240-minute movie time in one SMRTcell™.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Pacbio reads\u0026nbsp;were assembled using HGAP3 from SMRTportal (PacBio, CA, USA) with default settings.\u003c/p\u003e\n\u003cp\u003eFor NF_6988, barcoding and library preparation were performed using the Native Barcoding Kit 24 V14, followed by sequencing on an R10.4 flow cell in a MinION Mk1c device (Oxford Nanopore, Oxford, UK).\u0026nbsp;Base calling and adapter trimming were performed with Guppy version 6.3.8+d9e0f64 (Oxford Nanopore) using the super-accuracy configuration (dna_r10.4.1_e8.2_260bps_sup). The resulting reads were then assembled using Flye v. 2.9.2-b1786 \u003csup\u003e70\u003c/sup\u003e and inspected using Bandage \u003csup\u003e71\u003c/sup\u003e. Long-read polishing was performed using Medaka v. 1.0.3 (https://github.com/nanoporetech/medaka).\u0026nbsp;Both genomes were then annotated with Prokka 1.14.6\u0026nbsp;\u003csup\u003e72\u003c/sup\u003e before being submitted as complete genomes to NCBI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColicin production assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial overnight cultures were centrifuged at 3,000xg for 5 minutes and the culture medium supernatant was passed through a 0.2 µm filter to make spent media. Overnight cultures of test strains were diluted 1:100 in M9 minimal medium, mixed with sterile-filtered spent medium at 1:25 ratio and incubated for 4 h at 37°C before measuring the optical density at 600 nm. Spent media were prepared from \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eR12 wildtype, ∆colicin Y, and ∆colicins. Test strains were \u003cem\u003eE. coli\u003c/em\u003e R12 mutants lacking the colicins and corresponding immunities, or \u003cem\u003eE. coli\u003c/em\u003e K12 MG1655 lacking \u003cem\u003efepA\u003c/em\u003e, \u003cem\u003efhuA\u003c/em\u003e, \u003cem\u003eompA\u003c/em\u003e, alone or in combinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompetition assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of inhibitor and target cells were mixed at a ratio of 10:1 and 20 µl were spotted onto solid M9Gly+CAA minimal media. The cells were co-cultured for 24 h at 37°C before suspended in 1xPBS and plated onto LB solid media containing appropriate antibiotics to enumerate the number of colony-forming units per milliliter of inhibitors and targets. Competitive indices were calculated as the ratio of inhibitors to target cells at the end of the co-culture divided by the ratio at the start of the co-culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty-eight 7-week old female Balb/c (Charles River laboratories distributed by Scanbur) were housed in individual ventilated cages (Tecniplast boxunsfue) at the Swedish National\u0026nbsp;\u003cem\u003eVeterinary Institute\u003c/em\u003e. The mice were housed 8 mice per cage in a controlled environment (23°C\u0026nbsp;±\u0026nbsp;2, 75 ACH and 12h light and dark cycle). The mice’s diet was given an autoclaved pellet feed with no less than 18% protein, 5% fat and 5% fibre, tap water and the cages was enriched with autoclaved hay, a house and a tunnel. All personal handling the mice were required to change clothes, shoes and wear overall, gloves and cap before entering the room, also gloves were changed between cages. All the mice were handled with non-aversive tunnel handling since it has been shown to reduce stress and anxiety in laboratory mice \u003csup\u003e73\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter a seven-day acclamation period the mice\u0026nbsp;were split into 4 mice per cage before the start of the experiment. \u0026nbsp;For all inoculums, a single colony of each bacterial strain was grown in LB overnight. The gavage solution was prepared by diluting the overnight culture 1:10 in 1x Phosphate Buffered Saline (PBS) with bicarbonate solution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the experiment with undisturbed microbiota, mice were orally gavaged with 100\u0026nbsp;ml \u003cem\u003eE. coli\u003c/em\u003e R12 wildtype or ∆complete (10\u003csup\u003e7\u003c/sup\u003e CFU/mouse).\u0026nbsp;To ensure the bacteria did not affect the mice in any negative way the mice were closely watched after treatment and weighed every day to ensure no significant amount of weight were lost.\u0026nbsp;Fecal samples were taken for CFU counts on days -5, -2, 0-9 as well as DNA extraction and metagenomic sequencing on days 0 and 8.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the pre-colonization experiment, mice were given 5 g l\u003csup\u003e-1\u003c/sup\u003e streptomycin in drinking water for 7 days. At day -2 p.i., mice were treated with 20 mg streptomycin per mouse by gavage. \u0026nbsp; At day -1 p.i. streptomycin was removed from the water and mice were orally gavaged with \u003cem\u003eE. coli\u003c/em\u003e K12 MG1655 (10\u003csup\u003e7\u003c/sup\u003e colony-forming units (CFU)). The following day, challenger bacteria, \u003cem\u003eE. coli\u003c/em\u003e R12 wildtype, ∆CDI, ∆colicins or ∆complete were introduced by oral gavage (10\u003csup\u003e7\u003c/sup\u003e CFU). Fecal samples were taken for CFU counts on days -5, -2, 0-9.\u003c/p\u003e\n\u003cp\u003eTo determine the colony counts, each day fecal samples were obtained from each mouse. Fecal samples were weighed and resuspended in PBS using a plunger. The samples were serially diluted, plated on MacConkey agar supplemented with appropriate antibiotics, and CFU were determined by colony counting. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiolog PM metabolic phenotype analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phenotypic analysis of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eMG1655, \u003cem\u003eE. coli\u003c/em\u003e R12 and \u003cem\u003eE. coli\u003c/em\u003e NF_6988 were conducted using Biolog Phenotype Microarray (PM) plates, PM1 and PM2, following the recommended protocol\u0026nbsp;(Biolog Inc., Hayward, CA, USA). In summary, the bacteria were inoculated in LB\u0026nbsp;liquid media and incubated for 16 h prior to diluting the sample 1:35 in IF-0 to 42%T (OD\u003csub\u003e600\u003c/sub\u003e ~0.17) and subsequently diluted in IF-0+Dye to 85%T (OD\u003csub\u003e600\u003c/sub\u003e ~0.03). 100 µl per well were loaded on PM1 and P2 metabolic plates and the plates were incubated at 37°C, with orbital shaking, in an Infinite M200 microplate reader (Tecan). Absorbance at 590 nm was measured every 15 minutes for 48 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics\u003c/u\u003e\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with Swedish animal welfare laws. All procedures were done according to LASAs good practice guidelines and handling was done with non-aversive tunnel handling to improve animal welfare \u003csup\u003e73\u003c/sup\u003e. \u0026nbsp;The Uppsala Animal Experiments Ethics review board in Uppsala, Sweden approved all mice protocols undertaken in this study under reference no. 5.8.18-5552/2019.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw data for the manuscript are freely available either as supplementary excel files (Tables S2 and S3) or at the NCBI data repository for genome sequences (accession numbers are found in the text).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP.M., D.L., and S.K. conceived the study. P.M., E.K., and S. K. performed experiments. P.M., J.K, E.K., and S.K analyzed data. P.M., D.L., and S.K. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by grants from the Swedish research council, the foundation to prevent antibiotic resistance and Uppsala antibiotics center to S.K. We thank Mia Phillipson and Susan Schlegel for insightful input during the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrestoff, J.R. \u0026amp; Artis, D. 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Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice. \u003cem\u003eSci Rep\u003c/em\u003e 9, 20305 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9649958/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9649958/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe gut microbiome is essential for human health. Although the gut microbiota is largely stable at the species level in healthy individuals, strain-level variation remains less understood. Many bacterial strains encode toxin delivery systems that may shape competition within the gut. Here, we investigate how contact-dependent growth inhibition (CDI) and colicins influence intestinal colonization by a competitive murine \u003cem\u003eEscherichia coli\u003c/em\u003e isolate, R12. We show that R12 can colonize an intact mouse gut microbiota by displacing resident \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, but success depends on multiple interacting factors. CDI systems and colicins provide a competitive advantage against resident \u003cem\u003eE. coli\u003c/em\u003e, particularly during early colonization, while metabolic flexibility and access to alternative carbon sources support long-term persistence. Colonization outcomes vary between hosts and are shaped by resident microbiota composition, strain-level competition, and the initial invader-to-resident ratio. Overall, successful gut invasion is determined by the combined effects of bacterial antagonistic systems, metabolic capacity, and ecological context.\u003c/p\u003e","manuscriptTitle":"Roles of bacterial growth competition systems in colonization of the murine gut","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 07:04:24","doi":"10.21203/rs.3.rs-9649958/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"72ec6abe-4192-4861-b834-146af40d6458","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"checksComplete","content":"","date":"2026-05-10T23:05:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbiome","date":"2026-05-08T06:47:33+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T07:04:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 07:04:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9649958","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9649958","identity":"rs-9649958","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}