Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut

Abstract

The microbial communities that reside within the intestinal tract in vertebrates are complex and dynamic. In this report, we establish the utility of Caenorhabditis elegans as a model system for identifying the factors that contribute to bacterial persistence and for host control of gut luminal populations. We found that for N2 worms grown on mixed lawns of bacteria, Salmonella enterica serovar Typhimurium substantially outcompeted Escherichia coli, even when E. coli was initially present at 100-fold-higher concentrations. To address whether innate immunity affects the competition, the daf-2 and daf-16 mutants were studied; their total gut bacterial levels reflect overall capacity for colonization, but Salmonella outcompeted E. coli to an extent similar to wild-type worms. To address the role of virulence properties, Salmonella Δspi-1 Δspi-2 was used to compete with E. coli. The net differential was significantly less than that for wild-type Salmonella; thus, spi-1 spi-2 encodes C. elegans colonization factors. An E. coli strain with repeated in vivo passage had an enhanced ability to compete against an in vitro-passed E. coli strain and against Salmonella. Our data provide evidence of active competition for colonization niches in the C. elegans gut, as determined by bacterial factors and subject to in vivo selection.

Fig 1

Survival and density of colonizing bacteria in the C. elegans gut. (A) Survival of the C. elegans N2 strain and daf-2, daf-16, and phm-2 mutants when grown on lawns of E. coli OP50. (B) Load of E. coli OP50 within the gut of wild-type C. elegans N2 or in the daf-2, daf-16, and phm-2 mutants for the first 8 days after the L4 stage.

Fig 2

Resilience of bacterial gut colonization depends on the bacterial strain and on C. elegans age and genotype. Studies were done at 48 h (day 2) (A and B) or at 96 h (day 4) (C and D) following shift and competition assays, as described in Materials and Methods. C. elegans N2 or mutant strains were grown on lawns of the founder bacteria E. coli pSMC21 and then transferred to new plates seeded with S. Typhimurium pGB5 at 48 h (A) or at 96 h (C). Alternatively, C. elegans N2 or mutants were grown on lawns of the founder bacteria S. Typhimurium pSMC21 and then transferred to new plates seeded with E. coli pGB5 at 48 h (B) or at 96 h (D). Bacterial density was determined by plating the intestinal contents of lysed worms on selective media. Data represent means ± standard deviations (SD). Asterisks indicate a significant difference (P < 0.05) compared to the density of the founder strain recovered from N2 worms (*) or significant difference compared to the density of the introduced strain (**).

Fig 3

Effect of aging on E. coli founder strain persistence in C. elegans. C. elegans N2 worms were initially grown on lawns of founder E. coli DY330 (black bars) and then moved at different points in worm maturation (L4 stage plus 2, 3, 4, or 7 days) to plates with lawns of introduced E. coli OP50. Bars show intestinal densities of founder E. coli DY330 (black) and introduced E. coli OP50 (gray bars) recovered 24 h after shifting the worms to lawns of the introduced strain. The older the worms, the more the founder (DY330) strain was able to resist the newly introduced strains; with aging, the concentrations of the introduced (OP50) strain also increased but less dramatically. Data represent means ± SD.

Fig 4

E. coli OP50 can resist the intrusion of Salmonella in the gut of older worms. (A) Gut densities of E. coli OP50 (black bars) and S. Typhimurium SL1344 (gray bars) recovered from C. elegans N2 after their growth on founder E. coli OP50 lawns and then after the substitution of S. Typhimurium lawns at several points in worm maturation (L4 stage plus 1, 2, 3, 4, or 5 days). Data represent means ± SD. (B) Intestinal bacteria recovered from C. elegans N2 after being switched from lawns of founder E. coli OP50 (black bars) to lawns of S. Typhimurium SL1344 (gray bars), which were transferred on day 3 (L4 plus 3), with worms then maintained on the introduced S. Typhimurium strain with sampling of the worms at 24, 48, and 96 h after the shift.

Fig 5

In vitro growth and competition of E. coli and Salmonella. (A) Growth of E. coli, S. Typhimurium SL1344, and the SL1344 spi-1 spi-2 mutant on LB broth supplemented with 60 μg/ml streptomycin and incubated at 25°C; (B) competition between E. coli and S. Typhimurium SL1344 in vitro in broth culture; (C) competition between E. coli OP50 and the SL1344 spi-1 spi-2 mutant, as described in panel B.

Fig 6

Simultaneous competition between E. coli and S. Typhimurium for gut colonization of C. elegans N2 and mutants. (A) Gut densities at 24 h after exposure of N2 worms to S. Typhimurium SL1344-pRZT3 expressing DsRed (dark bars) and S. Typhimurium SL1344-pGB5 expressing GFP (gray bars) grown for 6 ho on mixed lawns at ratios of 10:1,000, 10:10, and 10:0.1. Data represent means ± SD. (B) Lawns with differing ratios (10:1,000, 10:10, 10:0.1) of E. coli OP50 and S. Typhimurium SL1344 were prepared, and worms fed on these lawns for 6 h. Levels of E. coli (black bars) and S. Typhimurium (gray bars) in the gut were measured 24 h later in wild-type C. elegans N2, as described above. (C) Fluorescence microscopy of C. elegans N2 after feeding for 12 h on mixed lawns of E. coli OP50-pGB5 and S. Typhimurium SL1344-pRZT3 at a ratio of 10:0.1 (100-fold E. coli excess). Arrows demarcate the gut lumen. Arrowheads indicate the pharyngeal grinder. Magnification, ×40. The green fluorescence channel shows E. coli inside the gut lumen and gut autofluorescence, and the red channel shows Salmonella fluorescence. The merged image shows bacterial colocalization (yellow). (D) Same as panel B in daf-2 mutants. (E) Same as panel B in daf-16 mutants.

Fig 7

Simultaneous competition between two variants of E. coli (A) and two variants of S. Typhimurium (B) for gut colonization of C. elegans N2. (A) Gut densities at 24 h after exposure of N2 worms to E. coli pSMC21 expressing Amp and Km resistance (black bars) and E. coli pGB5 expressing Amp and Tet resistance (gray bars) grown for 6 h on mixed lawns at ratios of 10:1,000, 10:10, and 10:0.1. Data represent means ± SD. (B) S. Typhimurium pSMC21 and pGB5 on lawns, as explained for panel A.

Fig 8

Role of the SPI-1/-2 islands in Salmonella colonization, resilience, and C. elegans longevity. (A) Life span assays of C. elegans raised on lawns of E. coli OP50 (black circles), wild-type S. Typhimurium SL1344 (gray diamonds), or S. Typhimurium SL1344 Δspi-1 Δspi-2 (white squares). (B) For worms grown on lawns of E. coli OP50 (dark bars), S. Typhimurium SL1344 (gray bars), or SL1344 Δspi-1 Δspi-2 (white bars), their gut densities in C. elegans N2 at L4 stage (L4 plus 0), day 4 (L4 plus 4), or day 8 (L4 plus 8) of their adult life span. Data represent means ± SD. An asterisk indicates a significant difference (P < 0.05) compared to growth on E. coli OP50. (C) Gut density of the S. Typhimurium SL1344 wild type (WT) (gray bars) and S. Typhimurium SL1344 Δspi-1 Δspi-2 (white bars), as described in panel D. (D) Gut density of E. coli OP50 (black bars) and S. Typhimurium SL1344 (gray bars) within wild-type C. elegans N2 grown on mixed lawns with ratios of 10:1,000, 10:10, and 10:0.1 of E. coli to Salmonella. Data represent means ± SD. (E) Gut density of E. coli OP50 (black bars) and S. Typhimurium SL1344 Δspi-1 Δspi-2 (white bars), as described in panel B.

Fig 9

Selection for bacteria with improved colonization. (A) Growth of E. coli and Salmonella cells in vitro at 25°C, as shown in Fig. 4A. Cells include E. coli in vivo passed for 10 generations of worms (black-and-white circles), E. coli with parallel in vitro passage (black circles), and wild-type S. Typhimurium SL1344 (gray triangles). Lawns with differing ratios (10:1,000, 10:10, 10:0.1) of in vitro-passed E. coli OP50 and in vitro-passed S. Typhimurium SL1344 were prepared, and 3-day-old worms (B) or 5-day-old worms (D) fed on these for 6 h. Gut levels of E. coli (black bars) and S. Typhimurium (gray bars) were measured 24 h later in wild-type C. elegans N2. An asterisk indicates a significant difference (P < 0.05) compared to panel B. E. coli OP50 was sequentially passed through the worm gut 10 times (see Materials and Methods). Lawns with differing ratios (10:1,000, 10:10, 10:0.1) of worm-passed E. coli OP50 and in vitro-passed S. Typhimurium SL1344 were prepared, and 3-day-old worms (C) or 5-day-old worms (E) fed on these for 6 h. Gut levels of E. coli (black-and-white bars) and S. Typhimurium (gray bars) were measured 24 h later in wild-type C. elegans N2. An asterisk indicates a significant difference (P < 0.05) compared to panel B. OD, optical density.