Flagella and Chemotaxis Are Required for Efficient Induction of Salmonella enterica Serovar Typhimurium Colitis in Streptomycin-Pretreated Mice

Bacterial strains and growth conditions.

Serovar Typhimurium strains were grown for 12 h at 37°C in Luria-Bertani broth containing 0.3 M NaCl, diluted 1:20 in fresh medium, and subcultured for 4 h with mild aeration. For infection experiments, bacteria were washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in cold PBS (5 × 10⁷ CFU/50 μl). For competitive infection experiments, identical volumes of subcultures were mixed prior to washing with PBS.

The serovar Typhimurium SL1344 mutants used in this study were constructed by phage transduction. To construct M913, the fliGHI::Tn10 allele was transduced from a lysate of SB245 (ΔsipA ΔiagP ΔsicP ΔsptP::aphT fliGHI::Tn10; K. Kaniga and J. E. Galan, unpublished data), and transductants were selected for growth on tetracycline. The fliGHI::Tn10 allele originated from X3420 (34). The chemotaxis mutant M935 was constructed by P22-mediated transduction of the cheY::Tn10 allele of SJW589 (45), which was kindly provided by Shigeru Yamaguchi.

SPI-1 mutant strains SB225 (sipA::aphT) (30), SB220 (sipC::aphT), SB169 (sipB::aphT) (30), and SB302 (invJ::aphT) (7) were used as control strains for analysis of supernatant proteins.

All strains were tested by stab inoculation of semisolid agar plates (10% tryptone, 5% NaCl, 0.3% agar) and incubation at 37°C for 5 to 8 h until bacterial halos that had radii of 1 to 2 cm were visible with wild-type Salmonella serovar Typhimurium strain SL1344. In addition, the motility of all strains was analyzed microscopically.

Construction of fluorescent protein expression plasmids.

Cloning of DNA fragments was performed by using standard protocols (39).

Plasmid pM979 (a pBR322 derivative) for expression of green fluorescent protein (GFP) under control of the constitutive rpsM promoter was constructed as follows. The coding sequence of GFPmut3b was amplified from pJBA27 (a kind gift from Christoph Jacobi) by PCR performed with primers 5′ GCA GAA TTC AGG AAA CAG TAT TCA TGC GTA AAG GAG AAG AAC TTT TC 3′ and 5′ CTG GAA TTC TTA TTT GTA TAG TTC ATC CAT GCC 3′ and was cloned into pBluescript (Stratagene) by using EcoRI to obtain pM900. pM935 was created by cloning GFPmut3b and its Shine-Dalgarno sequence by using XbaI and HindIII from pM900 into pWKS30 (50). The lac promoter was subsequently removed from pM935 by partially digesting it with BglII; this was followed by Acc651 digestion, filling in with the Klenow fragment, and religation to obtain pM946. pM958 was constructed by XbaI/BamHI digestion of pM946 and introduction of a fragment containing 536 bp upstream of rpsM (designated prpsM) amplified by PCR from SL1344 genomic DNA with primers 5′ CAT TCT AGA CGA TAA AGT AAT GAC CCG CCT 3′ and 5′ CAT GGA TCC GGG CCA CTA TGC ACT CCT ACT 3′. GFPmut2 was then PCR amplified from pKEN2 (9) with primers 5′ CAT GAA TTC AGG AGG TAG TAT TGA TGA GTA AAG GAG AAG AAC TTT 3′ and 5′ CAT GAA TTC GAA GAC TTA TTT GTA TAG TTC ATC CAT GCC 3′ and introduced into EcoRI-digested pM958, and the product was designated pM965. pM966 was created by subcloning GFPmut2 from pM965 into pBluescript by using EcoRI. GFPmut3b was cloned from pJBA27 into pM136 by using XbaI and HindIII (44) to obtain pM183. pM963 was constructed by removing sopE-GFP-M45 from pM183 by Eco47III/HindIII digestion and replacing it with prpsM-GFPmut3b (NotI/EcoRV) from pM958. prpsM-GFPmut3b was replaced in pM963 by GFPmut2 from pM966 by NotI/EcoRV digestion, and the resulting plasmid was designated pM968 (Table 1). The rpsM-promoter was recovered from pM963 by XbaI/BamHI digestion and introduced into pM968 to obtain pM979 (Table 1).

pDsRed, a pACYC184-derived plasmid, which expresses DsRed-Express (Clontech) under the control of the lac promoter, was a kind gift from Christoph Jacobi.

Animal experiments.

Animal experiments were performed at the BZL (Universität Zürich) as described previously (3) by using specific-pathogen-free female C57BL/6 mice that were 6 to 9 weeks old and were obtained from Harlan (Horst, The Netherlands). Water and food were withdrawn 4 h prior to per os (p.o.) treatment with 20 mg of streptomycin. After this, water and food were provided ad libitum. Twenty hours after the streptomycin treatment, water and food were withdrawn again for 4 h before the mice were infected with 5 × 10⁷ CFU of serovar Typhimurium (50 μl of a suspension in PBS p.o.). At the times postinfection (p.i.) indicated below, the mice were sacrificed by cervical dislocation, and tissue samples were removed from the intestinal tract, spleen, and liver for analysis. Animal experiments were approved by the Swiss authorities and were performed according to the legal requirements.

Analysis of serovar Typhimurium loads in the intestine, mLN, spleen, and liver.

To analyze colonization, the spleen, liver, and mesenteric lymph nodes (mLN) were removed aseptically and homogenized in PBS (containing 0.5% Tergitol and 0.5% bovine serum albumin) at 4°C as described previously (3). The bacterial loads were determined by plating samples on MacConkey agar plates containing 50 μg of streptomycin per ml. The minimal detectable level was 10 CFU/organ for the mLN, 20 CFU/organ for the spleen, and 100 CFU/organ for the liver.

Cecal contents were collected at different times p.i., and the bacterial loads were determined by plating. The minimum detectable level was 10 CFU per 25- to 150-mg sample of intestinal contents.

For coinfection experiments, the levels of colonization of mutant bacteria (carrying an appropriate antibiotic marker) and wild-type bacteria were determined by plating samples on MacConkey agar plates containing streptomycin or streptomycin and tetracycline. The values were confirmed by replica plating.

Histological procedures.

Tissue samples were embedded in O.C.T. (Sakura, Torrance, Calif.), snap frozen in liquid nitrogen, and stored at −80°C. Cryosections (5 μm) were mounted on glass slides, air dried for 2 h at room temperature, and stained with hematoxylin and eosin (HE).

Cecum pathology was independently evaluated by two pathologists in a blinded manner by using 5-μm-thick HE-stained sections and a histopathological scoring scheme described previously (3), as follows.

(i) Submucosal edema.

Submucosal edema (SE) was deduced from the extension of the submucosa, and values (expressed as percentages) were determined by morphometric analysis by using the following formula: SE = (b − a)/c, where a is the area enclosed by the mucosa (mucosa and intestinal lumen), b is the area enclosed by the borderline between the submucosa and tunica muscularis (submucosa, mucosa, and intestinal lumen), and c is the area enclosed by the outer edge of the tunica muscularis (tunica muscularis, submucosa, mucosa, and lumen; area of the whole cecal cross section). Submucosal edema was scored as follows: 0, no pathological changes; 1, detectable edema (submucosal edema, <10%); 2, moderate edema (submucosal edema, 10 to 40%); 3, profound edema (submucosal edema, ≥40%).

(ii) PMN infiltration into the lamina propria.

PMN in the lamina propria were enumerated by examining 10 high-power fields (magnification, ×400; field diameter, 420 μm), and the average number of PMN per high-power field was calculated. The results were scored as follows: 0, less than 5 PMN per high-power field; 1, 5 to 20 PMN per high-power field; 2, 21 to 60 PMN per high-power field; 3, 61 to 100 PMN per high-power field; 4, more than 100 PMN per high-power field.

(iii) Goblet cells.

The average number of goblet cells per high-power field (magnification, ×400) was calculated by examining 10 different regions of the cecal epithelium. A score of 0 indicated that there were more than 28 goblet cells per high-power field. In the cecum of the normal specific-pathogen-free mice we observed an average of 6.4 crypts per high-power field, and the average crypt consisted of 35 to 42 epithelial cells, 25 to 35% of which were differentiated into goblet cells. A score of 1 indicated that there were 11 to 28 goblet cells per high-power field; a score of 2 indicated that there were 1 to 10 goblet cells per high-power field; and a score of 3 indicated that there was less than 1 goblet cell per high-power field.

(iv) Epithelial integrity.

Epithelial integrity was scored as follows: 0, no pathological changes detectable in 10 high-power fields (magnification, ×400); 1, epithelial desquamation; 2, erosion of the epithelial surface (gaps of 1 to 10 epithelial cells per lesion); 3, epithelial ulceration (gaps of >10 epithelial cells per lesion; at this stage there was generally granulation tissue below the epithelium).

The combined pathological score for each tissue sample was determined by adding the averaged scores. The combined scores indicated the following conditions: 0, intestine intact without any signs of inflammation; 1 to 2, minimal signs of inflammation (frequently found in the ceca of specific-pathogen-free mice); 3 to 4, slight inflammation; 5 to 8, moderate inflammation; 9 to 13, profound inflammation.

Immunofluorescence experiments.

Streptomycin-pretreated animals were infected with 1:1 mixtures of wild-type and mutant bacteria carrying GFP or DsRed expression plasmids (pM979 and pDsred). Mice were killed on day 1 p.i., and cecal tissues were recovered and treated as described recently (4). Briefly, the tissues were fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4°C. The fixed tissue samples were washed with PBS and equilibrated in PBS containing 20% sucrose and 0.1% NaN₃ overnight at 4°C. The tissues were then embedded in O.C.T. (Sakura), snap frozen in liquid nitrogen, and stored at −80°C. Cryosections (7 μm) were mounted on glass slides and air dried for 2 h at room temperature prior to immunostaining. Sections were fixed in 4% paraformaldehyde for 5 min, washed, and blocked with 10% (wt/vol) normal goat serum in PBS for 1 h. The sections were stained for 1 h with polyclonal rabbit anti-Salmonella O-antigen group B serum (factors 1, 4, 5, and 12; 1:500 in PBS containing 10% [wt/vol] goat serum; Difco). Rhodamine-conjugated goat anti-rabbit serum (Dianova) diluted 1:300 in PBS containing 10% (wt/vol) goat serum was used as the secondary antibody. DNA was stained with DAPI (4′ 6′-diamidino-2-phenylindole) (0.5 μg/ml; Sigma). F-Actin was visualized by staining with Alexa-647-conjugated phalloidin (Molecular Probes). Sections were mounted with 50% glycerol in PBS, and each cover glass was sealed with nail polish.

The relative localization of GFP-positive and GFP-negative bacteria was determined as follows. Optical fields showing bacteria and epithelium were chosen for analysis. Images were recorded at a magnification of ×630 by using a Perkin-Elmer Ultraview confocal imaging system and a Zeiss Axiovert 200 microscope. Red, green, and cyan fluorescence was recorded confocally, while DAPI fluorescence was recorded by epifluorescence microscopy. Images were combined by using Adobe Photoshop, version 7.0.1. For quantification of GFP-positive bacteria close to the epithelium, all anti-LPS-stained (red) bacteria within 20 μm of the epithelium were counted. All GFP-positive bacteria that were LPS positive (green and red) were counted. The level of GFP-positive bacteria was expressed as a percentage of the total LPS-positive Salmonella population and was calculated as follows: number of GFP-positive red cells/number of red cells × 100.

Statistical analysis.

Statistical analyses of the individual pathological scores for submucosal edema, PMN infiltration, loss of goblet cells, and epithelial integrity and of the combined pathological score were performed by using the exact Mann-Whitney U test and the SPSS software, version 11.0, as described previously (3). P values of <0.05 were considered statistically significant. Bacterial colonization was analyzed in a similar manner. To allow statistical analysis of the bacterial loads, the values used for animals that yielded no CFU were the minimal detectable levels (mLN, 10 CFU; spleen, 20 CFU; liver, 100 CFU; intestinal contents, between 67 and 400 CFU [see above]). After this, the median values were calculated by using Microsoft Excel XP, and a statistical analysis was performed by using the exact Mann-Whitney U test and the SPSS software, version 11.0. P values of <0.05 were considered statistically significant.

Analysis of proteins from serovar Typhimurium culture supernatants.

Overnight cultures of serovar Typhimurium strains were grown in Luria-Bertani medium without antibiotics. Bacteria were pelleted by centrifugation at 8,000 × g for 20 min. Then 1.6 ml of each supernatant was filtered through 0.22-μm-pore-size low-protein-binding filters (Millex GV; Millipore). Proteins were precipitated for 2 h on ice by addition of 375 μl of trichloroacetic acid, followed by centrifugation at 14,000 × g for 30 min. Each precipitate was washed in ice-cold acetone and centrifuged. The resulting pellet was resuspended in sample buffer (50 mM Tris-Cl [pH 6.8], 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.1% bromphenol blue, 10% glycerol). The proteins were boiled for 5 min, separated on sodium dodecyl sulfate-10% polyacrylamide gels, and visualized with Coomassie brilliant blue R250 stain.

Preparation of chromosomal DNA and Southern blotting.

For Southern blot analysis chromosomal DNA of serovar Typhimurium strains were prepared by using standard methods (39). DNA was digested with EcoRV and HindIII, electrophoresed on a 1% agarose gel, and transferred to a ZETA-Probe BT blotting membrane with a vacuum blotter (Bio-Rad Laboratories). Southern hybridization was performed overnight at 58°C. Synthesis of fluorescein-labeled probes and detection were performed by using the protocols of the manufacturer (random prime labeling system, version II; Amersham, Little Chalfont, England). Probes used for detection of insertions in Salmonella genes fliGHI and cheY were amplified by PCR performed with primers fliGHI-fwd (5′GCC AGT GGA TGA GTA ACG AT 3′), fliGHI-rev (5′ CGT CGC CCT CCT GCT TTA T 3′), cheY-fwd (5′ TGC GCG TTA GTA AAG CTG GTA 3′), and cheY-rev (5′CCA GTC CGG CAG TGA TTA TTA 3′).

Construction and verification of flagellum mutants.

To construct an isogenic serovar Typhimurium mutant that lacked flagella, we transduced the fliGHI::Tn10 allele of SB245 into wild-type serovar Typhimurium strain SL1344. The resulting strain was designated M913. fliGHI code for class II genes of the flagellum regulon, the rotor/switch protein (fliG), the ATPase that drives flagellar export (fliI), and the negative regulator of fliI (fliH). Deletion of fliG is known to lead to a nonflagellated phenotype (24). Thus, M913 was nonmotile on motility agar (Table 1). Furthermore, M935, a mutant defective in chemotaxis, was created by P22 transduction of the cheY::Tn10 allele of SJW589 into SL1344. Correct insertion of the Tn10 insertion into the two mutants was verified by Southern blot hybridization by using probes specific for fliGHI and cheY, respectively (Fig. 1B). It is well established that flagellated serovar Typhimurium sheds a significant proportion of its flagellar subunits into the culture supernatant when it is grown in vitro. FliC (H1 flagellin; 52 kDa) is the most abundant secreted protein under these conditions (32). Therefore, we analyzed the secreted proteins of M913 and M935. To facilitate identification of flagellar subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several mutants with deletions in the SPI-1 TTSS apparatus (ΔinvG, invJ::aphT) and effector protein genes sipA, sipB and sipC were also included. As expected, M913 did export SPI-1 effector proteins but no flagellar subunits into the culture supernatant (Fig. 1A). Although an isogenic cheY::Tn10 mutant (M935) was unable to move in a chemotactic manner, it was able to assemble functional flagella (Table 1). These data confirmed that M913 and M935 could be used to assess the global importance of flagella and the specific role of chemotaxis in serovar Typhimurium colitis.

Mouse virulence of a nonflagellated mutant 2 days p.i.

First we investigated the role of flagella in murine serovar Typhimurium colitis. Two groups of five streptomycin-pretreated C57BL/6 mice were infected intragastrically with 5 × 10⁷ CFU of mutant M913 lacking flagella or wild-type strain SL1344. At day 2 p.i., the animals were killed, and we analyzed colonization of the cecum, spleen, liver, and mLN, as well as pathological changes in the cecal tissue. We found that the levels of colonization of the cecum and mLN by the two strains were not significantly different (Fig. 2A and B). The levels of mutant M913 were slightly but not significantly higher in the liver and spleen (P ≥ 0.05) (Fig. 2C and D). This finding is consistent with the findings of previous studies of the relevance of flagella for systemic infection in the murine typhoid model (40). Histopathological analysis revealed that there was a small but significant difference in the total pathological scores between M913 and the isogenic wild-type strain (Fig. 2E) (P = 0.016, as determined by the Mann-Whitney U test), but the differences between the scores for the individual parameters were not significant. The inflammation in mice infected with the nonflagellated strain appeared to be slightly weaker than the inflammation in mice infected with the wild-type strain 2 days p.i. even though the two groups of mice had equal numbers of bacteria in their cecal contents and mLN. This indicates that flagella may play a role in the initiation of colitis in streptomycin-pretreated mice.

Time course of cecum pathology.

At 2 days p.i., the colitis caused by a nonflagellated serovar Typhimurium strain was slightly attenuated. It has been shown that streptomycin-pretreated mice develop colitis as early as 8 h p.i. (3). This raised the possibility that flagella might play a more pronounced role in the early phase of infection. To test this hypothesis, we infected 15 streptomycin-pretreated mice with wild-type serovar Typhimurium and 15 streptomycin-pretreated mice with nonflagellated strain M913. At 10, 24, and 48 h p.i. five mice from each group were killed and analyzed as described above.

Both strains efficiently colonized the cecum (10⁸ to 10⁹ CFU/g) within 10 h p.i., and the level of colonization remained constant throughout the rest of the experiment (Fig. 3A). The level of colonization of the mLN of three of five animals infected with the wild-type serovar Typhimurium strain was low, while no bacteria were detected in the mLN of animals infected with nonflagellated mutant M913. However, the difference was not statistically significant (Fig. 3B). Moreover, no differences between colonization of the mLN by M913 and colonization of the mLN by wild-type serovar Typhimurium were observed at later times. In accordance with previous data (3), colonization of the spleen and liver was apparent between 24 and 48 h p.i. While we recovered slightly higher numbers of wild-type bacteria than of the nonmotile mutant, the differences were not statistically significant (Fig. 3C and D). In spite of the similar colonization levels, M913 caused significantly reduced levels of inflammation (Fig. 3E and Table 2). In keeping with our first observation, the difference was small but significant at 48 h p.i. but much more pronounced at earlier times (Fig. 3E). At 10 h p.i., the ceca of M913-infected mice showed almost no signs of inflammation, such as PMN influx, edema, or epithelial damage. In contrast, mice infected with wild-type serovar Typhimurium had already developed severe colitis at this time. At 24 h p.i., mice infected with the fliGHI mutant showed the first symptoms of inflammation, and at day 2 p.i., there was only a small difference between the pathological scores for the two groups. This implies that the onset of inflammation is delayed upon infection with a nonmotile serovar Typhimurium mutant.

Localization of the fliGHI mutant.

The experiments described above demonstrated that flagella are required for efficient induction of serovar Typhimurium colitis in streptomycin-pretreated mice. However, the bacterial loads in the intestinal lumen do not account for the difference observed. Therefore, we reasoned that less efficient penetration of the mucus layer and reduced access to the intestinal epithelium might provide a plausible explanation. To test this hypothesis, we performed competition experiments. In this type of experiment, streptomycin-pretreated animals are infected with a mixture of wild-type and mutant bacteria. If each strain is labeled with a different fluorescent marker (GFP or DsRed), fluorescence microscopy can be used to compare the relative capacities of the two strains to interact with the intestinal wall.

M913 and the isogenic wild-type strain were transformed with GFP (pM979) and DsRed (pDsRed) expression plasmids. Preliminary infection experiments confirmed that the plasmids were stably propagated by both strains in the murine intestine for at least 2 days. Less than 5% of all serovar Typhimurium cells recovered from the cecum contents had lost the plasmids (data not shown).

We then performed competitive infection experiments (Fig. 4). Three streptomycin-pretreated mice were infected with a 1:1 mixture of wild-type strain SL1344(pDsRed) and strain M913(pM979) (Fig. 4A). Three other mice were infected with a 1:1 mixture of wild-type strain SL1344(pM979) and strain M913(pDsRed) (Fig. 4B). At 24 h p.i., the animals were sacrificed, and cecum tissue samples were processed and stained for immunofluorescence microscopy (see Materials and Methods); DNA was stained with DAPI and was grey, and f-actin was stained with Alexa-647-phalloidin and was blue. As DsRed fluorescence was too weak for reliable detection, we stained all serovar Typhimurium cells in the cecum sections with a rabbit anti-LPS antibody and an anti-rabbit serum-rhodamine conjugate (which was red) (see Materials and Methods). In the first group of mice, red bacteria (wild type) were more abundant than yellow-green bacteria [M913(pM979)] at the epithelial surface (Fig. 4A). In contrast, roughly equal numbers of the competing strains were present in the cecal lumen (Fig. 4D). Very similar observations were made with the second group of mice; yellow-green bacteria (wild type) were more abundant than red bacteria [M913(pDsred)] at the epithelial surface (Fig. 4B), but equal numbers of the two strains were present in the cecal lumen (Fig. 4D). These findings indicate that flagella are required by serovar Typhimurium to efficiently contact the intestinal epithelium.

In order to quantify the defect in M913, we determined the numbers of wild-type and mutant bacteria present within 20 μm of the epithelial surface (Fig. 4C) (see Materials and Methods). The data were obtained by scoring at least 400 bacteria from different optical fields of cecum sections from all three mice from each group. The results were expressed as percentages of the GFP-positive red bacteria (number of GFP-positive red bacteria/number of red bacteria × 100). In this way we verified that the fliGHI mutant M913 was deficient in terms of approaching the cecal epithelium (Fig. 4D). The results of all of the control experiments performed were consistent with this conclusion (Fig. 4D). In mice infected with a 1:1 mixture of wild-type strain SL1344(pM979) and wild-type strain SL1344(pDsRed), roughly equal numbers of the two strains were present in the cecal lumen and close to the epithelial surface (Fig. 4D). Infections with wild-type SL1344(pM979) alone and infections with a strain carrying a control vector (pM968; no GFP expression) yielded >95% and <5% GFP-positive red bacteria, respectively, at the epithelium (Fig. 4D); >95% of both strains recovered from the intestinal lumen harbored the appropriate plasmid (Fig. 4D). These observations demonstrated that the GFP vector allowed reliable detection and discrimination between wild-type and mutant strains. Altogether, these data provide strong evidence that serovar Typhimurium requires flagella to approach the intestinal epithelium efficiently.

A chemotaxis mutant is attenuated for causing enterocolitis.

The data presented above demonstrated that the Salmonella mutant M913 lacking the entire flagellum secretion apparatus is impaired in terms of causing colitis and that this virulence defect may be linked to a reduced ability to approach the epithelial surface. In order to examine whether the virulence defect might be attributed to a chemotaxis defect, we constructed M935, a cheY transposon insertion mutant of serovar Typhimurium, by phage transduction (see Materials and Methods). This mutant was able to assemble functional flagella and secrete flagellin (FliC) (Fig. 1A), but it was not able to swim in a directed manner (45). On motility agar M935 formed no halo (Table 1). We performed two independent experiments with five streptomycin-pretreated mice per group. The animals were infected with 5 × 10⁷ CFU of serovar Typhimurium M913, M935, or wild-type strain SL1344, and we analyzed colonization and cecal inflammation 24 h p.i. The results were identical in both experiments and are summarized in Fig. 5. fliGHI mutant M913, cheY mutant M935, and the isogenic wild-type strain had the same capacity to colonize the cecal lumen and the mLN (Fig. 5A and B). No significant colonization of the liver and spleen was detected in any of the experimental groups. cheY mutant M935 and the nonflagellated mutant M913 induced significantly less inflammation than the wild-type strain induced. There was no significant difference in pathology between M913 and M935 (P ≥ 0.05) (Fig. 5E and Table 3). These data were consistent with the results described above and suggested that chemotactic movement through the mucus layer is required for efficient induction of colitis.

Competitive infection experiments to assess colonization defects of cheY and fliGHI mutants.

A second type of competition experiment was performed to examine whether motility and chemotaxis contribute to intestinal colonization in the streptomycin-pretreated mouse model. In the previous experiments we analyzed just one strain per mouse (Fig. 2, 3, and 5). This did not reveal any major colonization defect of nonmotile or nonchemotactic strains in the cecal lumen. However, a competition experiment could allow detection of even subtle defects. In this experiment, two groups of five streptomycin-pretreated mice were infected with a 1:1 mixture (total amount, 5 × 10⁷ CFU) of wild-type strain SL1344 and M913 or wild-type strain SL1344 and M935. The ratio of the two strains in the feces was analyzed at days 1 and 2 p.i. The mice were sacrificed at day 3 p.i., and we analyzed the bacterial loads and the ratios of the two strains in the cecal contents, mLN, spleen, and liver.

In the feces from each of the groups, equal numbers of the two strains were found at day 1 p.i. (competitive indices [CI], ∼0.3 to 1.2). The CI values for the nonmotile and nonchemotactic strains decreased at day 2 p.i., and the CI for M913 and M935 in the cecal contents at day 3 p.i. was significantly lower (median, 10⁻²) (Fig. 6A).

The median CI for M913 and M935 in the mLN, spleen, and liver ranged from 1 to 0.1 (Fig. 6B, C, and D). These data suggest that flagella and, more specifically, chemotaxis contribute to serovar Typhimurium colonization of the murine intestine but not to the spread to systemic sites.