Impaired fitness and transmission of macrolide-resistant Campylobacter jejuni in its natural host

Abstract

Campylobacter jejuni is a major zoonotic pathogen transmitted to humans via the food chain and is prevalent in chickens, a natural reservoir for this pathogenic organism. Due to the importance of macrolide antibiotics in clinical therapy of human campylobacteriosis, development of macrolide resistance in Campylobacter has become a concern for public health. To facilitate the control of macrolide-resistant Campylobacter, it is necessary to understand if macrolide resistance affects the fitness and transmission of Campylobacter in its natural host. In this study we conducted pairwise competitions and comingling experiments in chickens using clonally related and isogenic C. jejuni strains, which are either susceptible or resistant to erythromycin (Ery). In every competition pair, Ery-resistant (Ery(r)) Campylobacter was consistently outcompeted by the Ery-susceptible (Ery(s)) strain. In the comingling experiments, Ery(r) Campylobacter failed to transmit to chickens precolonized by Ery(s) Campylobacter, while isogenic Ery(s) Campylobacter was able to transmit to and establish dominance in chickens precolonized by Ery(r) Campylobacter. The fitness disadvantage was linked to the resistance-conferring mutations in the 23S rRNA. These findings clearly indicate that acquisition of macrolide resistance impairs the fitness and transmission of Campylobacter in chickens, suggesting that the prevalence of macrolide-resistant C. jejuni will likely decrease in the absence of antibiotic selection pressure.

Fig 1

In vitro growth kinetics of clonally related and isogenic C. jejuni strains. The cultures were incubated in MH broth at 42°C under microaerobic conditions with shaking (160 rpm). Each data point represents the mean log₁₀ CFU/ml of five technical replicates. The experiment was repeated twice and similar results were obtained. 700819, Bd34-2, and Bd41-3 are Ery susceptible, while J.L. 270, J.L.272, J.L.273, and T.L.102 are Ery resistant (see Table 1 for MIC values).

Fig 2

Colonization levels of Ery^s C. jejuni ATCC 700819 (●) and clonally related Ery^r strains J.L.272 (□) and J.L.273 (△) in chickens. Each Campylobacter strain was individually inoculated into chickens at the concentration of 1.4 × 10⁶ CFU/bird (ATCC 700819), 5.56 × 10⁵ CFU/bird (J.L.272), and 2.57 × 10⁵ CFU/bird (J.L.273). Fecal samples were collected at 3, 6, and 10 days after inoculation. Each data point represents the number of Campylobacter obtained from an individual chicken. The mean colonization level (log₁₀ CFU/g feces) of each group is indicated by a horizontal bar.

Fig 3

Pairwise competition between Ery^s and Ery^r Campylobacter in chickens in the absence of antibiotic selection pressure. (A to E) Competition between clonally related isolates. (A) ATCC 700819 (●) versus J.L.270 (○); (B) ATCC 700819 (●) versus J.L.272 (□); (C) ATCC 700819 (●) versus J.L.273 (△); (D) Bd34-2 (■) versus J.L.272 (□); (E) Bd41-3 (▲) versus J.L.273 (△). (F to H) Competition between isogenic strains. (F) ATCC 700819 (●) versus T.L.101 (◐); (G) ATCC 700819 (●) versus T.L.102 (◧); (H) ATCC 700819 (●) versus T.L.103 (◭). Each symbol represents the number of Ery^s or Ery^r Campylobacter in an individual chicken. The horizontal bars represent the mean colonization levels (log₁₀ CFU/g feces) of Ery^s or Ery^r strains detected at each sampling time point.

Fig 4

Levels of Campylobacter colonization in chickens before and after comingling. (A) Colonization levels of Campylobacter in chickens (n = 8) precolonized with Ery^s C. jejuni ATCC 700819 before and after comingling with chickens (n = 4) precolonized with Ery^r transformant T.L.101. The numbers of 700819 and T.L.101 in the chickens are indicated by solid circles (●) and open triangles (△), respectively. (B) Colonization levels of Campylobacter in chickens (n = 4) precolonized with Ery^r transformant T.L.101 before and after comingling with chickens (n = 8) precolonized with Ery^s C. jejuni ATCC 700819. The numbers of 700819 and T.L.101 in the chickens are indicated by by solid circles (●) and open triangles (△), respectively. (C) Colonization levels of Campylobacter in chickens (n = 5) precolonized with Ery^s C. jejuni ATCC 700819 before and after comingling with chickens (n = 11) precolonized with Ery^r transformant T.L.102. The numbers of 700819 and T.L.102 in the chickens are indicated by solid circles (●) and open diamonds (♢), respectively. (D) Colonization levels of Campylobacter in chickens (n = 11) precolonized with Ery^r transformant T.L.102 before and after comingling with chickens (n = 5) precolonized with Ery^s C. jejuni ATCC 700819. The numbers of 700819 and T.L.102 in the chickens are indicated by solid circles (●) and open diamonds (♢), respectively. (E) Colonization levels of Ery^r strain T.L.101 in chickens (n = 8) in the absence of competing Ery^s C. jejuni. These non-mingled chickens were used as a control for the comingling study. In panels A to E, each data point represents the log₁₀ transformed CFU number/g of feces from a single bird, and the mean colonization level (log10 CFU/g of feces) is indicated by a horizontal bar.