Enhanced biofilm formation and multi-host transmission evolve from divergent genetic backgrounds in Campylobacter jejuni

Abstract

Multicellular biofilms are an ancient bacterial adaptation that offers a protective environment for survival in hostile habitats. In microaerophilic organisms such as Campylobacter, biofilms play a key role in transmission to humans as the bacteria are exposed to atmospheric oxygen concentrations when leaving the reservoir host gut. Genetic determinants of biofilm formation differ between species, but little is known about how strains of the same species achieve the biofilm phenotype with different genetic backgrounds. Our approach combines genome-wide association studies with traditional microbiology techniques to investigate the genetic basis of biofilm formation in 102 Campylobacter jejuni isolates. We quantified biofilm formation among the isolates and identified hotspots of genetic variation in homologous sequences that correspond to variation in biofilm phenotypes. Thirteen genes demonstrated a statistically robust association including those involved in adhesion, motility, glycosylation, capsule production and oxidative stress. The genes associated with biofilm formation were different in the host generalist ST-21 and ST-45 clonal complexes, which are frequently isolated from multiple host species and clinical samples. This suggests the evolution of enhanced biofilm from different genetic backgrounds and a possible role in colonization of multiple hosts and transmission to humans.

Figure 1

Genetic relatedness and biofilm formation of C . jejuni and C . coli isolates. Population structure of (A) C . jejuni and (B) C . coli with each isolate labelled as belonging to the upper (red; OD ₆₀₀ above 0.272), middle (pink; OD₆₀₀ between 0.201 and 0.272) or lowest (white; OD₆₀₀ below 0.201) 33rd percentile of biofilm formers based upon phenotype assays. MLST clonal complexes coloured according to the common isolate source for C . jejuni generalists (black), cattle specialists (blue), chicken specialists (orange) and wild bird specialists (green). Trees were based on 761 381 and 1 018 234 bp core genome alignments for C . jejuni and C . coli, respectively, calculated using an approximation of the maximum likelihood algorithm. The scale bar represents the average number of polymorphisms per site. Local support values on each branch were calculated with default values in FastTree2. Values reflect the reliability of each split in the tree and were estimated using the Shimodaira–Hasegawa test on the three alternate topologies around that split, at a default re‐sampling rate of 1000 re‐samples for each split. (C) Biofilm formation scores in four major ecological groups of C ampylobacter including host generalists, chicken specialists, cattle specialists and C . coli. The proportion of isolates in the upper, middle and lowest biofilm formation percentile in these four ecological groups is statistically different (χ² test; P = 0.0116).

Figure 2

Genome position of biofilm‐associated genetic elements. A. Frequency of biofilm‐associated 30 bp words from the original and/or verification genome‐wide association studies, mapped to the C . jejuni  NCTC11168 reference genome for ST‐21 (blue) and ST‐45 (red) clonal complex isolates. B. Visualized using Artemis (Carver et al., 2009) showing biofilm‐associated words (inner circle) in relation to annotated coding regions (black lines) and oxidative stress (orange), capsule and extrapolysaccharides (purple), nickel transport (green) and biofilm genes from published studies (Svensson et al., 2009; Howlett et al., 2012; Avila‐Ramirez et al., 2013; van Alphen et al., 2014).

Figure 3

Correlation of biofilm formation with oxidative stress resistance phenotypes. A. Distribution of H ₂ O ₂ inhibition zone diameters (cm) on agar plates for isolates in the upper (above OD ₆₀₀ of 0.272), middle and lowest (below OD ₆₀₀ of 0.201) 33rd percentile of biofilm formation. Each point represents at least three biological replicates per isolate. The mean is indicated, with error bars representing the standard deviation. B. Distribution of biofilm formation (OD at 600 nm) at different oxygen concentrations (20% – atmospheric, 10% and 5%). Tukey box plots are shown, the horizontal bar represents the median and the box encompasses half of all data points for the corresponding condition. Asterisks in both panels indicate statistical significant differences between conditions with one asterisk for P ≤ 0.05, two for P ≤ 0.01 and three for P ≤ 0.001.