Campylobacter jejuni strains compete for colonization in broiler chicks

Abstract

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal(r) strain) and one streptomycin-resistant strain (the 02-833L Str(r) strain). In vitro binding assays revealed that the C. jejuni F38011 Nal(r) and 02-833L Str(r) strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str(r) strain relative to that of the C. jejuni F38011 Nal(r) strain competitively inhibited the binding of the C. jejuni F38011 Nal(r) strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str(r) strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal(r) strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.

FIG. 1.

Assessment of C. jejuni motility on MH medium supplemented with 0.4% agar. C. jejuni F38011 was isolated from a human with bloody diarrhea, and C. jejuni 02-833L was isolated from the carcass of a chicken. The C. jejuni CS strain, which was recovered from a chick, is a naturally occurring nonmotile isolate. Exponential-phase cultures were inoculated to motility plates and incubated as described in Materials and Methods.

FIG. 2.

Representative growth curves of C. jejuni F38011 Nal^r and 02-833L Str^r strains. MH broth was inoculated with the C. jejuni F38011 Nal^r and 02-833L Str^r strains at an OD₅₄₀ of 0.02 and incubated under microaerobic conditions with constant shaking (60 rpm) at 37°C. Cultures are indicated as follows: C. jejuni F38011 Nal^r alone (open squares), C. jejuni 02-833L Str^r alone (open triangles), and C. jejuni F38011 Nal^r (closed squares) cocultured with C. jejuni 02-833L Str^r (closed triangles).

FIG. 3.

Adherence of the C. jejuni F38011 Nal^r and 02-833L Str^r strains to LMH chicken hepatocellular carcinoma epithelial cells. Binding assays were performed as outlined in Materials and Methods, with a 5.0 × 10⁷-CFU inoculum of each strain. “Mix” refers to LMH cells inoculated with equal doses of the C. jejuni F38011 Nal^r and 02-833L Str^r strains. Each bar represents the mean ± standard deviation for C. jejuni F38011 Nal^r (open bars) and C. jejuni 02-833L Str^r (gray bars) bound to the LMH cells per well of a 24-well plate. The difference in binding between strains for the mixed inoculation was determined to be statistically significant (P < 0.05).

FIG. 4.

IF microscopy showing the peripheral association of the C. jejuni F38011 Nal^r (A) and 02-833L Str^r (B) strains to LMH chicken hepatocellular carcinoma epithelial cells. LMH cell-associated C. jejuni bacteria were stained with a rabbit anti-C. jejuni antibody, followed by incubation with a Cy2-conjugated donkey anti-rabbit immunoglobulin G antibody. Actin (red staining) was stained using tetramethylrhodamine isothiocyanate-labeled phalloidin. Cell nuclei (blue) were stained with DAPI.

FIG. 5.

Competitive inhibition of the binding of the C. jejuni F38011 Nal^r strain to LMH chicken hepatocellular carcinoma epithelial cells with the C. jejuni 02-833L Str^r strain. Binding assays were performed as outlined in Materials and Methods, with a 1.7 × 10⁷-CFU inoculum of the C. jejuni F38011 Nal^r strain. A 2.9 × 10⁹-CFU inoculum of C. jejuni 02-833L Str^r represents a 170-fold increase for the competitor strain. Each bar represents the mean ± standard deviation for C. jejuni F38011 Nal^r (open bars), C. jejuni 02-833L Str^r (gray bars), and S. enterica serovar Typhimurium (filled bars) bound to the LMH cells per well of a 24-well plate. An asterisk indicates that the value is significantly different from that of the control (P < 0.05).

FIG. 6.

Excess C. jejuni 02-833L Str^r reduces the binding of the C. jejuni F38011 Nal^r strain to LMH cells, but an excess in the number of C. jejuni F38011 Nal^r bacteria does not reduce the binding of C. jejuni 02-833L Str^r to LMH cells. Binding assays were performed as outlined in Materials and Methods. In the left panel, a constant-level inoculation with 2.0 × 10⁶ CFU of the C. jejuni F38011 Nal^r strain was challenged with up to 4.2 × 10⁹ CFU of the C. jejuni 02-833L Str^r strain, representing a 2,100-fold increase for the competitor strain. In the right panel, a constant-level inoculation with 4.2 × 10⁶ CFU of the C. jejuni 02-833L Str^r strain was challenged with up to 2.0 × 10⁹ CFU of the C. jejuni F38011 Nal^r strain, representing a 475-fold increase for the competitor strain. Each bar represents the mean ± standard deviation for C. jejuni F38011 Nal^r (open bars) and C. jejuni 02-833L Str^r (gray bars) bound to the LMH cells per well of a 24-well plate. An asterisk indicates that the value is significantly different from that of the control (P < 0.05).

FIG. 7.

C. jejuni 02-833L reduces the efficiency of intestinal colonization by C. jejuni F38011 in broiler chicks. C. jejuni F38011 and C. jejuni 02-833L were recovered from the intestinal tracts of chicks at 14 dpi as outlined in Materials and Methods. The circle indicates uninoculated chicks. Each square indicates the number of viable C. jejuni F38011 Nal^r bacteria recovered from a pooled intestinal sample. Each triangle indicates the number of viable C. jejuni 02-833L Str^r bacteria recovered from a pooled intestinal sample. n indicates the number of birds in the group of 10 from which no viable C. jejuni bacteria were recovered (limit of detection, 10³ CFU/gram intestinal contents). The bar indicates the mean number of bacteria that were recovered from only those birds that were colonized with C. jejuni.

FIG. 8.

C. jejuni 02-833L reduces the efficiency of cecal colonization by C. jejuni F38011 in broiler chicks. C. jejuni F38011 and C. jejuni 02-833L were recovered from the ceca of chicks at 14 dpi as outlined in Materials and Methods. The circle indicates uninoculated chicks. Each square indicates the number of viable C. jejuni F38011 Nal^r bacteria recovered from a cecum. Each triangle indicates the number of viable C. jejuni 02-833L Str^r bacteria recovered from a cecum. n indicates the number of birds in the group of 10 from which no viable C. jejuni bacteria were recovered (limit of detection, 10³ CFU/gram cecal contents). The bar indicates the mean number of bacteria that were recovered from only those birds that were colonized with C. jejuni.