Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae

Abstract

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of ~100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.

Fig. 1.

Blooms of commensal E. coli in S. Tm infected mice. (A) Colonization levels of commensal E. coli in cecal contents of S. Tm infected mice. E. coli CFU were detected by routine screening on MacConkey without antibiotics by colony morphology and lactose-positive phenotype. Pie plots on the right show microbiota composition in three mice (marked in green, red, and blue). Here, total DNA was extracted, and bacterial 16S rRNA genes were PCR-amplified using universal bacterial primers, cloned, and sequenced (∼100 sequences per animal). (B) DNA microarray comparing the virulence gene content of 10 E. coli isolates collected from S. Tm infected mice from 2002 to 2007 (indicated in red) and different pathogenic and nonpathogenic E. coli reference strains. Genes were grouped with the CLUSTER software based on the presence (red) or absence (black) of genes. Groups of genes belonging to distinct islands or phages are indicated. (C) Coinfection experiments of S. Tm and different E. coli strains. Streptomycin-treated, E. coli-free mice were coinfected with 1:1 mixtures of wild type S. Tm and E. coli strains Ec^MG1655, or the ECOR B2 strains Ec^Nissle, Ec⁸¹⁷⁸ and Ec^CFT073 (total of 5 × 10⁷; 1:1; intragastrically). E. coli (white) and S. Tm (black) colonization density in the cecum at day 4 p.i. (Log₁₀ cfu/g). Bars show the median.

Fig. 2.

Highly similar conjugative plasmid is present in S. Tm and commensal E. coli. (A) S. Tm and Ec⁸¹⁷⁸ share a nearly identical conjugative plasmid. Sequence comparison of p2 S. Tm (outer circle) and p2 Ec⁸¹⁷⁸ (inner circle); gene functions are color coded. (B) Phylogram showing relation of p2 to other R-64 and ColIb-P9-type conjugative plasmids (E. coli PEK104, S. Tm R64-1, S. Tm R64-2, S. Heidelberg pSL476, Shigella sonnei pColb-P9, S. Tm pNF1358, S. Kentucky CVM29188, E. coli SE11-1). The phylogram was generated using PHYLIB and the Clustal W multiple sequence alignment algorithm; branch length represents the relative nucleotide differences of each plasmid compared with the reference sequence p2. (C) p2 is detected by gel electrophoresis in 4 of 10 E. coli isolates collected from S. Tm-infected mice (from Fig. 1B).

Fig. 3.

Conjugative Salmonella plasmid can confer a fitness benefit and is efficiently transferred to commensal E. coli in vivo. (A) Colicin Ib production leads to superior growth of S. Tm in competition against Ec^Nissle in the gut. Streptomycin-treated mice were coinfected with 1:1 mixtures (total of 5 × 10⁷; 1:1; intragastrically) of Ec^Nissle and S. Tm^wt or S. Tm^wt p2^Δcib, respectively. Transfer of p2 into Ec^Nissle could not be detected, which is in line with the established resistance against HGT of this strain. B., C. P2 is transferred to Ec^8178_cured by conjugation with high efficiency. Streptomycin-treated mice were coinfected with 1:1 mixtures (total of 5 × 10⁷; 1:1; intragastrically) of Ec^8178_cured and S. Tm^wt carrying p2^kan or p2^ΔoriTnikA, respectively. Densities of the S. Tm donor (black) and E. coli recipient strains (white) were determined in the feces at day 1 p.i. (B) and in the cecal contents at day 4 p.i. (C), and frequency of kanamycin-resistant E. coli transconjugants in cecal contents was determined by selective plating at day 4 p.i. (D). E. coli (white) and Salmonella (black) colonization densities (Log₁₀ cfu/gram) were determined in the feces at day 1 p.i. (B) or cecum content at day 4 p.i. (A and C). Bars show the median.

Fig. 4.

Inflammation boosts conjugative p2 transfer by increasing density of donors and recipients. (A) Streptomycin-treated low complexity microbiota (LCM; Left) or conventional (CON; Center and Right) mice were coinfected with 1:1 mixtures (total of 5 × 10⁷; 1:1; intragastrically) of Ec^8178_cured and S. Tm^wt or S. Tm^avir strains carrying p2^cm, respectively. Densities of the S. Tm donor and E. coli recipient strains were determined in the cecal contents at day 4 p.i. (B) The frequency of chloramphenicol-resistant E. coli transconjugants in cecal contents was determined by selective plating at day 4 p.i. Bars show the median. (C) Competing gut microbiota hinders bacterial contact as shown by immunofluorescent staining of Ec⁸¹⁷⁸ and S. Tm in the cecal lumen of a representative mouse of the experiment shown in a. Serial sections of PFA-fixed cecum tissue were stained with α-E. coli O7 (recolored blue) or α-S. Tm O4.5 antiserum (recolored magenta). Microbiota and host nuclear DNA was stained with Sytox green (recolored yellow). Arrows point at granulocytes in the cecal lumen of mice with gut inflammation. (Scale bar: 10 μm.)

Fig. 5.

Intestinal density of S. Tm plasmid donors correlates with E. coli transconjugant frequency. Streptomycin-treated conventional mice (n = 20) were coinfected with mixtures of Ec^{8178_cured amp} (recipient; amp^R), S. Tm^tet p2^Δcib (donor; tet^R; plasmid p2: kan^R) and increasing amounts of S. Tm^wt p2^{ΔoriTnikAΔcib} (competitor; cm^R) at no (n = 4), 10-fold (n = 3), 100-fold (n = 3), 1,000-fold (n = 3), 10,000-fold (n = 3), and 100,000-fold (n = 4) excess over the donor strain (total of 5 × 10⁷; intragastrically). The density of the S. Tm donor (x axis) was plotted against E. coli transconjugant frequencies (fraction of amp^R kan^R E. coli of total amp^R E. coli; y axis) as determined in the feces at day 1 p.i. (A) and cecal contents at day 4 p.i. (B). A linear relationship between cfu and transconjugant frequency could be predicted by linear regression analysis (P < 0.05).