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. 2010 Jan 1;201(1):71-80.
doi: 10.1086/648616.

Analysis of Acquisition of Pseudomonas Aeruginosa Gastrointestinal Mucosal Colonization and Horizontal Transmission in a Murine Model

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Free PMC article

Analysis of Acquisition of Pseudomonas Aeruginosa Gastrointestinal Mucosal Colonization and Horizontal Transmission in a Murine Model

Akinobu Kamei et al. J Infect Dis. .
Free PMC article

Abstract

Background: Laboratory systems to study bacterial transmission and mucosal colonization leading to infection have not been utilized.

Methods: We determined whether transmission of various strains of Pseudomonas aeruginosa among individual mice could occur and investigated the properties of such strains in establishing gastrointestinal (GI) mucosal colonization as well as in disseminating systemically after induction of neutropenia.

Results: P. aeruginosa isolates associated with epidemic spread among patients with cystic fibrosis (CF) readily established GI colonization at higher levels than did strains associated with acute infections in patients without CF, and they outcompeted these strains. Colonization was associated with resistance to bile salts. However, epidemic CF isolates did not disseminate after induction of neutropenia and did not induce as much mucosal pathology as did strains that were capable of disseminating.

Conclusion: Murine models can be used to study P. aeruginosa transmission and early colonization, and the properties of these strains associated with their known clinical behaviors are mimicked in this setting.

Figures

Figure 1
Figure 1
Gastrointestinal colonization levels in C3H/HeN mice by individual P. aeruginosa strains PAO1, PA14, PA2192nm, LES, or C3719 on day 1 (A) and day 5 (B) after the initiation of oral bacterial administration. Points represent results from individual animals, and horizontal lines represent the medians (n = 8 per strain). * P<0.01 compared to the non-epidemic strains of PAO1, PA14 and PA2192nm, ** P<0.01 compared to strain PA14. P values: Mann-Whitney test with Bonferroni's correction. (C), Bile salt sensitivities of P. aeruginosa strains PAO1, PAO1ΔgalU, PA14, PA2192nm, LES and C3719. Percent survival was defined as the number of CFUs counted after exposure to 1% sodium deoxycholate for 4 h at 37°C divided by CFUs after exposure to 2% BSA (control) in the same condition X 100. The bars show the average of triplicate samples for each strain, and the error bars represent standard error of the means. # P<0.01 compared to strain PAO1ΔgalU by one-way ANOVA with Dunnett's multiple comparison. (D), Serum sensitivities of P. aeruginosa strains PAO1, PAO1ΔgalU, PA14, PA2192nm, LES and C3719. Percent survival was defined as the number of CFUs counted after exposure to 10% human serum for 1 h at 37°C divided by CFUs after exposure to heat inactivated serum in the same condition X 100. The bars show the average of triplicate samples for each strain, and the error bars represent standard error of the means. Note Y axis is a log scale. Ψ P<0.01 compared to strain PA2192nm by one-way ANOVA with Dunnett's multiple comparison.
Figure 2
Figure 2
(A), (B), (C), (D), (E), (F); Gastrointestinal (GI) co-colonization levels by P. aeruginosa strains in individual mice 5 days after being given a mixture of one epidemic strain (LES or C3719) and one non-epidemic strain (PAO1, PA14 or PA2192nm) (n = 8 mice per each combination of strains). Colonization levels achieved by the epidemic strains were higher than those by the non-epidemic strains ((A), (B), (C), (D), (E), * P<0.05 by Wilcoxon signed rank test) except for one combination of strains C3719 and PAO1 (F). (G), Competitive Indices (C.I.) of GI co-colonization levels by epidemic versus non-epidemic strains in each mouse in the different groups. C.I. was defined as the colonization level of an epidemic strain divided by the level of a non-epidemic strain. A C.I. above 1.0 indicates the epidemic strain out-competed the non-epidemic strain in the co-colonization level. Horizontal bars indicate median, error bars indicate interquartile range.
Figure 3
Figure 3
Horizontal transmission of P. aeruginosa strains PAO1 (A), PA14 (B), PA2129nm (C), LES (D), and C3719 (E) from a previously colonized C3H/HeN mouse (○) to antibiotic-treated non-colonized mice (△, ■, ×) within a single cage. Points represent results from individual animals. All the P. aeruginosa strains were transmitted from a single colonized mouse to 3 antibiotic-treated uncolonized cage mates. Colonization levels in the gastrointestinal tract established an equilibrium, increasing until days 2 or 3 and then decreasing by day 5 for all the strains. P. aeruginosa was not detected in the acidified drinking water provided in the cages.
Figure 4
Figure 4
(A), Survival curves of C3H/HeN mice mono-colonized with P. aeruginosa strain PAO1, PA14, PA2192nm, LES, or C3719 followed by MAb RB6-8C5 induced neutropenia (n = 8 per strain). * P< 0.01, ** P<0.05 compared to mice colonized with strain PAO1 or PA14, # P<0.05 compared to those with strain PA2192nm by log rank test with Bonferroni's correction. (B), Bacterial levels of P. aeruginosa strains isolated from the spleens of moribund or dead mice after gastrointestinal colonization followed by the induction of neutropenia (n = 7, 8, and 5 for strain PAO1, PA14, and PA2192nm respectively). Ψ P<0.05 by Mann Whitney test with Bonferonni's correction.
Figure 5
Figure 5
Gross anatomy and microscopic histology of the murine (C3H/HeN) gastrointestinal (GI) tract after prior mono-colonization with P. aeruginosa strain PA14 ((A), (C), (E)) or LES ((B), (D), (F)) and subsequent MAb RB6-8C5 induced neutropenia. Mice were sacrificed and sections were obtained 42 h after induction of neutropenia. (A), Diffuse erythema in noncontiguous areas of the GI tract, notably in the small intestine as well as the tip of the cecum. (C), (E); Shortening and flattening of the villi along with marked sloughing of the epithelial cells are clearly seen. (B), (D), (F); Normal macro- and microscopic appearances. Magnification: (C), (D) 100X, (E), (F) 400X.
Figure 6
Figure 6
Levels of Tumor Necrosis Factor alpha (TNF-α) (A), and Interleukin one beta (IL-1ß) (B) in the ceca of C3H/HeN mice whose gastrointestinal tract were mono-colonized by either P. aeruginosa strain PA14 (n = 8) or strain LES (n = 6), followed by MAb RB6-8C5 induced neutropenia. Mice that were antibiotic-treated but not given any P. aeruginosa were also induced neutropenic to serve as non-colonized controls (n = 4). Mice were sacrificed 42 h after MAb administration and ceca harvested to measure the tissue cytokine levels. Lines indicate medians, boxes 25th and 75th percentiles, error bars 10th and 90th percentiles. * P<0.05 by Kruskal-Wallis test with Dunn's multiple comparison post-hoc test.

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