Competing isogenic Campylobacter strains exhibit variable population structures in vivo

Abstract

Consumption of poultry contaminated with Campylobacter jejuni is a risk factor for human gastrointestinal disease. The rational development of control strategies for Campylobacter within chickens requires an understanding of the colonization process at the molecular and population levels, both within and between hosts. Experiments employing competing strains of Campylobacter have been used to investigate colonization. Implicit in these studies is the assumption that the behavior of competing strains is reproducible between experiments. Variability in the recovery of mutants from the chicken gastrointestinal tract during signature-tagged mutagenesis studies demonstrated that this is not always the case. To further investigate this phenomenon in the absence of confounding factors due to phenotypic differences between mutants, we constructed individually identifiable wild-type isogenic tagged strains (WITS) that have indistinguishable phenotypes in pure culture. By using mixtures of WITS, it is possible to monitor the relative amounts of subpopulations of essentially wild-type bacteria. Using a 2-week-old chicken model of colonization, we observed unpredictable variations in population structure both within and between experiments, even in the simplest case of two competing strains. This variation occurred both when birds were simultaneously infected with two WITS and when birds inoculated with different WITS were cohoused. We present evidence for founder effects during initial colonization with subsequent bird-to-bird transmission. We suggest that these and phenotypic variation contribute to the observed variability. These factors render simple models of colonization which do not take them into account inappropriate for Campylobacter and impact the planning and interpretation of competition experiments using this organism.

FIG. 1.

Population structure of WITS during competition and transmission experiments. Data represent the total cecal loads of WITS and relative amounts of WITS CC001a and CC003a postmortem in the ceca of birds at the indicated days postinfection. The predominating strains are shown on the axes. Red rectangles, bias in favor of CC001a (log₁₀ CI of >0); blue rectangles, bias in favor of CC003a (log₁₀ CI of <0). The width of the rectangles corresponds to 95% confidence intervals. Where only one strain was detected, this is denoted by a filled circle. nd, not done; nde, no detectable colonization; vs, versus.

FIG. 2.

Population structure of WITS in the ceca of birds following inoculation with a 1:1 mix of WITS at a range of total doses. Data represent the cecal loads of WITS and CI of CC001b versus CC003a in individual birds at days 1, 3, and 7 postinoculation with a 1:1 (vol/vol) mix of the two strains at the indicated doses. The predominating strains are shown on the axes. Red rectangles, bias in favor of CC001b (log₁₀ CI of >0); blue rectangles, bias in favor of CC003a (log₁₀ CI of <0). The width of the rectangles corresponds to 95% confidence intervals. Where only one strain was detected, this is denoted by a filled circle. nd: not done; ndc: no detectable colonization; vs, versus.

FIG. 3.

Population structure of WITS in the ceca of birds following inoculation with a 1:1 mix of WITS at a range of total doses. Data represent the cecal loads of WITS and CI of CC001b versus CC003a in individual birds at days 1, 3, and 7 postinoculation with a 1:1 (vol/vol) mix of the two strains at the indicated doses. The predominating strains are shown on the axes. Red rectangles, bias in favor of CC001b (log₁₀ CI of >0); blue rectangles, bias in favor of CC003a (log₁₀ CI of <0). The width of the rectangles corresponds to 95% confidence intervals. Where only one strain was detected, this is denoted by a filled circle. ndc: no detectable colonization; vs, versus.