Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competition

Abstract

Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol—a nutrient that supports the growth of only E. coli—to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.

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Fig. 1: Streptomycin causes stochastic changes in the microbiota composition and a decrease in diversity in singly housed mice compared with co-housed mice.
Fig. 2: Co-housed mice are resistant to E. coli colonization, in contrast to most singly housed mice.
Fig. 3: K. michiganensis provides colonization resistance against E. coli.
Fig. 4: Competition for simple sugars provides a fitness advantage to K. michiganensis over E. coli.
Fig. 5: A nutritional advantage for E. coli abolishes the colonization-resistance capacity of K. michiganensis.
Fig. 6: K. michiganensis delays gut expansion of S. Typhimurium.

Data availability

The K. michiganensis (ARO112) Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number WMDR00000000. The version described in this paper is version WMDR01000000. The 16S rRNA gene sequencing data (Illumina sequences) obtained in this study are available at the Sequence Read Archive (SRA) NCBI database under BioProject ID PRJNA590204. Source data are available for Figs. 1–6 and Extended Data Figs. 1–7.

Code availability

The code used for Illumina sequencing analyses in this study is available at https://github.com/mothur/mothur, with modifications that are freely available from the corresponding author on request. MATLAB routines used for data visualization are available from the corresponding author on request.

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Acknowledgements

We thank T. Sana, E. Cascales and M. Blokesch for helpful discussions; J. Xavier, C. Ubeda and M. Taga for suggestions and for reading the manuscript; S. Higginbottom for help with mouse experiments; and R. Balbontín-Soria for providing strain RB249. We acknowledge support from the Allen Discovery Center at Stanford on Systems Modeling of Infection (to K.M.N. and K.C.H.); the Portuguese national funding agency Fundação para a Ciência e Tecnologia (FCT) PTDC/BIA-MIC/4188/14 and research infrastructure ONEIDA and CONGENTO projects (LISBOA-01-0145-FEDER-016417 and LISBOA-01-0145-FEDER-022170) co-funded by Fundos Europeus Estruturais e de Investimento from Programa Operacional Regional Lisboa 2020 (to R.A.O. and K.B.X.). K.B.X., R.A.O. and V.C. acknowledge the FCT for individual grants IF/00831/2015, PD/BD/106000/2014 and SFRH/BPD/116806/2016, respectively. J.L.S. and K.C.H. are Chan Zuckerberg Biohub Investigators.

Author information

R.A.O. performed and analysed streptomycin treatment and gnotobiotic mice experiments with assistance from V.C. and supervision from K.B.X. K.M.N. performed ciprofloxacin-treatment mouse experiments, with supervision by K.C.H. and J.L.S. R.A.O. and K.M.N. performed microbiota analyses. R.A.O., M.B.C. and V.C. performed bacterial growth curves. H.S. performed and analysed microfluidics experiments and single-cell imaging. R.A.O. and V.C. prepared tissue sections and K.M.N. performed imaging analyses. R.A.O., K.C.H. and K.B.X. designed the study and wrote the original draft of the paper. All of the other authors contributed to writing the manuscript.

Correspondence to Karina B. Xavier.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Streptomycin treatment leads to increased loss of core OTUs in singly housed mice compared to co-housed mice, but does not differentially affect Bacteroidetes, Firmicutes, or Verrucomicrobia relative abundances.

ac, Relative abundances of the a, Bacteroidetes, b, Firmicutes and c, Verrucomicrobia phyla in co-housed and singly housed mice at indicated time points. Data shown are medians, and error bars show the interquartile range of n=15-21 per group. d, Percentage of core OTUs (defined as OTUs present in every mouse of each group before treatment) present on days 15 and 57, relative to day 0. Data from ac represent n=20 co-housed mice and n=21 singly housed mice from 4 independent experiments. Data in d represent n=3 cages (day 15) or n=4 cages (day 57) of co-housed mice and n=21 (day 15) or n= 15 (day 57) singly housed mice from 3 (day 15) or 4 (day 57) independent experiments. Lines in ad represent medians and error bars depict interquartile ranges. Data in a-c were analyzed with two-tailed Mann-Whitney test (**: p<0.01; ns, not significant). For d, data were analyzed using the one-way Kruskal-Wallis test with Dunn’s correction test for multiple comparisons (*: q < 0.1, **: q < 0.05; ****: q < 0.001; ns, not significant). Source data

Extended Data Fig. 2 Ciprofloxacin treatment results in stochastic extinction of the Bacteroidetes phylum in singly housed mice.

a, Experimental scheme for ciprofloxacin treatment, from one experiment. Three milligrams of ciprofloxacin were administered orally every 12 h to conventional Swiss Webster mice for 5 days. Microbiota composition was analyzed from fecal samples collected from day 0 (before antibiotics), day 5 (last day of antibiotic treatment), and day 14 (9 days after stopping antibiotic treatment). Procedures (circles) denote the start (day 0) or the end (day 5) of ciprofloxacin treatment, and sacrifice mice (day 14). b, Fecal microbiota compositions on day 0, 5, and 14 of co-housed (Mouse 1-5) and singly housed (Mouse 6-11) mice. Each stacked bar represents the microbiota composition in the indicated mouse at the indicated time points. The colored segments represent the relative fraction of each genus-level taxon present at >3%. All other genera were combined in the “Other Bacteria” category. c, Phylogenetic dissimilarities on each day determined by the mean weighted Unifrac distance of the bacterial communities of each mouse to each other mouse within the same group. Data shown are medians, and the error bars show interquartile ranges (*: p<0.05; ns: not significant; two-tailed Mann-Whitney test, n=5 mice in the co-housed group, n=6 mice in the singly housed group). d, Gamma diversity of gut microbiota of co-housed and singly housed mice at indicated time points (n=1 cage in co-housed group, n=5 mice in singly housed group). Source data

Extended Data Fig. 3 Effects of streptomycin treatment on microbiota composition and OTUs associated with colonization resistance to E. coli in cohort 2 (associated with the experiments shown in Figs. 1 and 2).

a, Fecal microbiota compositions of untreated (day 0), streptomycin-treated (day 15), and post-streptomycin-treated (day 57) samples from co-housed and singly housed mice in cohort 2. Each stacked bar represents the microbiota composition in one mouse at the indicated time points. Colored segments represent the relative fraction of each genus-level bacterial taxon present at >3%. All other genera were combined into the “Other Bacteria” category. b, Histogram of linear discriminant analysis (LDA) scores >2 for differentially abundant OTUs on day 57 of mice in cohort 2 from the experiment in Fig. 2c, d. Red designates OTUs enriched in mice with colonization resistance to E. coli (CR); green designates OTUs enriched in mice lacking colonization resistance (No CR). c, Relative abundances of significantly different individual OTUs that are associated with lack of colonization resistance against E. coli. d, Relative abundances of significantly different individual OTUs that are associated with colonization resistance against E. coli. e, Area Under the Curve (AUC) calculated from the dynamics of E. coli-YFP CFUs/g feces during the cohort 2 experiment of each mouse with and without colonization resistance shows that Klebsiella-associated mice had significantly lower loads of E. coli throughout the experiment. Data in be represent n=6 CR mice and n=4 No CR mice from 1 experiment. In ce, medians and interquartile ranges are shown. In b, LEfSe uses the Kruskal-Wallis rank-sum test on a normalized relative abundance matrix to detect significantly different OTU features and estimates the effect size of each feature via LDA; only features with LDA scores >2 and alpha<0.05 were considered different between the groups of mice and are shown. Asterisks (*) in b represent statistical results from c and d. Data in c and d were analyzed with two-tailed Wilcoxon test using a Benjamini-Hochberg correction for multiple comparisons (: q<0.1). Data in e were analyzed with two-tailed Mann-Whitney test (**: p<0.01). Source data

Extended Data Fig. 4 K. michiganensis and E. coli-CFP colonization loads from experiment in Fig. 3c–e.

a, Loads of K. michiganensis (CFUs/g feces) before and after challenge with E. coli-YFP (n=9 across two independent experiments, Fig. 3d). b, Area Under the Curve (AUC) calculated from the dynamics of E. coli-YFP CFUs/g feces of each mouse with and without K. michiganensis. Mice associated with K. michiganensis had significantly lower loads of E. coli throughout the experiment (n=9 across two independent experiments, Fig. 3d). c, K. michiganensis (n=11 across three independent experiments) and E. coli-CFP (n=8 across two independent experiments) loads (CFUs/g feces) in mice pre-colonized with E. coli-YFP (Fig. 3c, e). d, AUC calculated from the dynamics of E. coli-YFP CFUs/g feces of each mouse after challenge with K. michiganensis or E. coli-CFP. Mice challenged with K. michiganensis had significantly lower loads of E. coli throughout the experiment (n=11 across three independent experiments) as compared to challenge with E. coli-CFP (n=8 across two independent experiments; Fig. 3e). In a and c, circles and lines represent values from individual mice and medians, respectively. In b and d, medians and interquartile ranges are shown. Data in b and d were analyzed with the two-tailed Mann-Whitney test (***: p<0.001; ****: p<0.0001). Source data

Extended Data Fig. 5 No killing of E. coli or direct physical inhibition by K. michiganensis was observed in vitro.

a, Co-cultures of E. coli-YFP with E. coli-CFP or K. michiganensis in minimal media containing 0.25% of the indicated carbon source exhibited similar percentages of PI-stained cells, suggesting the absence of active killing of E. coli. b, There was no statistically significant difference in the growth rates of E. coli microcolonies that were well separated from versus surrounded by K. michiganensis cells over >50% of the periphery in minimal medium with arabinose. c, Cell density of E. coli-YFP in the bottom chamber of a transwell incubated with K. michiganensis or E. coli-CFP in the upper chamber in minimal media with arabinose or fructose. Growth was monitored by OD600 measurements for 35 h (n=3 per condition). d, Cell density of E. coli-YFP in minimal medium with fructose with or without supplementation of 20% cell-free spent medium from overnight cultures of K. michiganensis or E. coli-YFP+K. michiganensis grown in minimal medium with fructose. Growth was monitored by OD600 measurements for 25 h (n=3 per condition). Data in b were analyzed with two-tailed Mann-Whitney test (ns: not significant). Lines and error bars in bd represent means and standard deviations, respectively. Source data

Extended Data Fig. 6 Galactitol can sustain growth of E. coli-YFP but not K. michiganensis in vitro; E. coli-YFP colonization of a singly housed mouse in which Klebsiella spp. were not eliminated by streptomycin treatment, and loads of the 2nd colonizer from the experiment shown in Fig. 5.

a, Growth capacity of K. michiganensis, E. coli-YFP, and E. coli-YFP ΔgatZ alone in minimal medium containing 0.25% galactitol. Growth was monitored by OD600 measurements at the indicated times for 43 h (n=6 per condition). K. michiganensis and E. coli-YFP ΔgatZ were unable to grow on galactitol, by contrast to E. coli-YFP. b, While measuring the capacity of galactitol to affect K. michiganensis-mediated colonization resistance against E. coli in singly housed mice after streptomycin treatment, we noted that one mouse presented high levels of resident Klebsiella spp. In this mouse, E. coli-YFP loads (CFUs/g feces) decreased immediately after gavage (day -3) (yellow solid line). This mouse was not included in the data displayed in Fig. 5b (see Supplementary Discussion). These data are from n=1 mouse from one experiment. Dashed yellow line shows the median CFUs/g feces of E. coli-YFP from Fig. 5b for comparison. c, Yellow line shows K. michiganensis loads (CFUs/g feces) in mice pre-colonized with E. coli-YFP and drinking water supplemented with 2% galactitol (n=7 across two independent experiments, Fig. 5a). Black dashed line shows median CFUs/g feces of K. michiganensis from the experiment in Fig. 2c, e for comparison (Extended Data Fig. 4c). d, Area Under the Curve (AUC) calculated from the dynamics of E. coli-YFP CFUs/g feces of each mouse drinking galactitol-supplemented water after challenge with K. michiganensis (n=7 across two independent experiments, Fig. 5b) compared with dynamics of E. coli-YFP CFUs/g feces of each mouse drinking non-supplemented water with and without K. michiganensis (data from Extended Data Fig. 4d) . Mice drinking galactitol-supplemented water had significantly higher E. coli loads throughout the experiment when compared with mice drinking non-supplemented water, even in the presence of K. michiganensis. e, K. michiganensis loads (CFUs/g feces) in ex-germ-free mice pre-colonized with E. coli-YFP or E. coli-YFP ΔgatZ drinking non-supplemented or 2% galactitol-supplemented water (n=6 per condition across two independent experiments, Fig. 5c). f, AUC calculated from the dynamics of E. coli-YFP CFUs/g feces of each ex-germ-free mouse drinking non-supplemented or 2% galactitol-supplemented water after challenge with K. michiganensis (n=6 per group across two independent experiments, Fig. 5d). Mice drinking galactitol-supplemented water had significantly higher E. coli-YFP loads throughout the experiment when compared with mice drinking non-supplemented water or E. coli-YFP ΔgatZ in mice drinking galactitol-supplemented water, even in the presence of K. michiganensis. Data in a represent means and standard deviations. In c and e, circles and lines represent values from individual mice and medians, respectively. In d and f, medians and interquartile ranges are shown. In d and f, data were analyzed using the Kruskal-Wallis test with Dunn’s correction test for multiple comparison (*: q<0.1; ***: q<0.01; ns, not significant). Source data

Extended Data Fig. 7 Competition for simple sugars by K. michiganensis and commensal E. coli with S. Typhimurium, and colonization loads of K. michiganensis and commensal E. coli from the experiment in Fig. 6.

a, S. Typhimurium-mCherry growth capacity in co-cultures with K. michiganensis, commensal E. coli or an isogenic S. Typhimurium (without a fluorescent-protein marker) in minimal media containing 0.25% of the indicated carbon source. S. Typhimurium-mCherry growth was monitored by mCherry fluorescence quantification (n=6 per condition). b, Area Under the Curve (AUC) calculated from the dynamics of S. Typhimurium CFUs/g feces of each mouse in the first 3 days after S. Typhimurium challenge in mice pre-colonized with K. michiganensis (n=9 across 2 independent experiments), as compared to mice not pre-colonized or pre-colonized with a commensal E. coli (n=8 across 2 independent experiments and n=4 in 1 experiment, respectively; Fig. 6). Mice colonized with K. michiganensis had significantly lower S. Typhimurium loads as compared to mice not pre-colonized or pre-colonized with a commensal E. coli. c, Loads of K. michiganensis (n=9 mice across two independent experiments) and commensal E. coli (n=4 mice, from one experiment) in CFUs/g feces before and after challenge with S. Typhimurium (Fig. 6b). d, Mice body weight from day 19 to 23 was unaffected by K. michiganensis (n=5 mice from one experiment), commensal E. coli (n=4 mice from one experiment) colonization, similarly to non-challenged. In a, lines and error bars represent means and standard deviations, respectively. In b, lines and errors bars represent medians and interquartile ranges, respectively. Data in b were analyzed using the one-sided Kruskal-Wallis test with Dunn’s correction test for multiple comparisons (**: q < 0.05; ns, not significant). In c and d, circles and lines represent values from individual mice and medians, respectively. Source data

Supplementary information

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Supplementary Fig. 1, Supplementary Table 1 and Supplementary Discussion.

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Statistical source data table for Supplementary Fig. 1.

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 4

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Source Data Extended Data Fig. 5

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Source Data Extended Data Fig. 6

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Source Data Extended Data Fig. 7

Statistical Source Data table for Extended Data Fig. 7.

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Oliveira, R.A., Ng, K.M., Correia, M.B. et al. Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competition. Nat Microbiol (2020). https://doi.org/10.1038/s41564-019-0658-4

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