Competitive Exclusion Prevents Colonization and Compartmentalization Reduces Transmission of ESBL-Producing Escherichia coli in Broilers

Abstract

Extended spectrum beta-lactamase (ESBL)-producing bacteria are resistant to extended-spectrum cephalosporins and are common in broilers. Interventions are needed to reduce the prevalence of ESBL-producing bacteria in the broiler production pyramid. This study investigated two different interventions. The effect of a prolonged supply of competitive exclusion (CE) product and compartmentalization on colonization and transmission, after challenge with a low dose of ESBL-producing Escherichia coli, in broilers kept under semi-field conditions, were examined. One-day-old broilers (Ross 308) (n = 400) were housed in four experimental rooms, subdivided in one seeder (S/C1)-pen and eight contact (C2)-pens. In two rooms, CE product was supplied from day 0 to 7. At day 5, seeder-broilers were inoculated with E. coli strain carrying bla _{CTX-M- 1} on plasmid IncI1 (CTX-M-1-E. coli). Presence of CTX-M-1-E. coli was determined using cloacal swabs (day 5-21 daily) and cecal samples (day 21). Time until colonization and cecal excretion (log₁₀ CFU/g) were analyzed using survival analysis and linear regression. Transmission coefficients within and between pens were estimated using maximum likelihood. The microbiota composition was assessed by 16S ribosomal RNA gene amplicon sequencing in cecal content of broilers on days 5 and 21. None of the CE broilers was CTX-M-1-E. coli positive. In contrast, in the untreated rooms 187/200 of the broilers were CTX-M-1-E. coli positive at day 21. Broilers in C2-pens were colonized later than seeder-broilers (Time to event Ratio 3.53, 95% CI 3.14 to 3.93). The transmission coefficient between pens was lower than within pens (3.28 × 10^-4 day^-2, 95% CI 2.41 × 10^-4 to 4.32 × 10^-4 vs. 6.12 × 10^-2 day^-2, 95% CI 4.78 × 10^-2 to 7.64 × 10^-2). The alpha diversity of the cecal microbiota content was higher in CE broilers than in control broilers at days 5 and 21. The supply of a CE product from day 0 to 7 prevented colonization of CTX-M-1-E. coli after challenge at day 5, likely as a result of CE induced effects on the microbiota composition. Furthermore, compartmentalization reduced transmission rate between broilers. Therefore, a combination of compartmentalization and supply of a CE product may be a useful intervention to reduce transmission and prevent colonization of ESBL/pAmpC-producing bacteria in the broiler production pyramid.

Keywords: ESBL; Escherichia coli; antimicrobial resistance; colonization; compartments; intervention; poultry.

FIGURE 1

Schematic representation of the experimental set up of one of four broiler rooms (1–4). Each room was subdivided in nine pens, with one seeder (S/C1) pen in the middle (2 m²) (n = 10 S-broilers, 10 C1-broilers), surrounded by eight contact (C2) pens (1 m²) (n = 10 broilers per pen). The S/C1-pen was separated from the C2-pens by 80 cm high mesh panels. The C2-pens were separated from each other by 80 cm high wooden panels.

FIGURE 2

Time until colonization (days) of CTX-M-1-E. coli per pen (S/C1, C2₁, C2₂, C2₃, C2₄, C2₅, C2₆, C2₇, C2₈) and type of bird [seeder (S), contact 1 (C1), contact 2 (C2)] for room 1 (left) and room 2 (right). The violin plot indicates the total range of observations; the black dot indicates the median.

FIGURE 3

Alpha (phylogenetic) diversity of cecal microbiota at day 5 (n = 5 broilers per intervention) and day 21 (n = 40 broilers per intervention), for the control (rooms (R) 1, 2) and intervention groups (rooms (R) 3, 4).

FIGURE 4

Principal coordinate analysis (PCoA) of microbiota composition based on weighted UniFrac (A) and unweighted UniFrac (B) distances between control (dark blue) and CE (light blue) groups. Different symbols indicate different sampling days: triangles are samples of day 5, and circles are samples of day 21.

FIGURE 5

Heatmap representing the abundance of amplicon sequence variants (ASVs) in all individual broiler chickens analyzed (n = 90). Only ASVs that are significantly different at day 5 and day 21 between CE and control are shown (Wilcoxon rank-sum test, adjusted p-values are corrected p-values for multiple testing, Benjamini–Hochberg, p < 0.05). Each red, white, or blue rectangle represents the relative abundance of a genus in an individual broiler. Clustering of broilers is based on Ward’s minimum variance method and based on weighted UniFrac distances matrix.