Network analysis highlights complex interactions between pathogen, host and commensal microbiota

Abstract

Interactions between bacteria and their host represent a full continuum from pathogenicity to mutualism. From an evolutionary perspective, host-bacteria relationships are no longer considered a two-component system but rather a complex network. In this study, we focused on the relationship between brook charr (Salvelinus fontinalis) and bacterial communities developing on skin mucus. We hypothesized that stressful conditions such as those occurring in aquaculture production induce shifts in the bacterial community of healthy fish, thus allowing pathogens to cause infections. The results showed that fish skin mucus microbiota taxonomical structure is highly specific, its diversity being partly influenced by the surrounding water bacterial community. Two types of taxonomic co-variation patterns emerged across 121 contrasted communities' samples: one encompassing four genera well known for their probiotic properties, the other harboring five genera mostly associated with pathogen species. The homeostasis of fish bacterial community was extensively disturbed by induction of physiological stress in that both: 1) the abundance of probiotic-like bacteria decreased after stress exposure; and 2) pathogenic bacteria increased following stress exposure. This study provides further insights regarding the role of mutualistic bacteria as a primary host protection barrier.

Figure 1. Scheme of the study and sampling design.

The design comprise two different units (test vs control) composed of four tanks, all sharing the same water and biofilter. Fish were equally split between the two units and were raised in the same conditions. Only the fish from the test unit were exposed to stress. The sampling design is a 35 days protocol. On day 0, we sampled the time 0 bacterial communities from mucus, biofilters, biofilm and water. On day 7, the fish from the test unit were exposed to the stress. On day 14, 21, 28 and 35, we sampled the bacterial communities (samples time 1, 2, 3 and 4 respectively) from mucus, biofilters, biofilm and water.

Figure 2. Plasma cortisol concentration in Salvelinus fontinalis before and after hypoxia exposure.

“Control” represents fish sampled in the control units; “test 1” represent fish sampled before stress exposure in the trial units; “test 2” represents fish sampled 10 min following stress exposure. Each point represents means from 80 fish measurements (10 individuals from 8 families). The presence of statistical significant differences (represented by a and b) was tested by a Wilcoxon rank pair-test (p < 0.001).

Figure 3. Relative abundance of different microbial taxonomic groups from different communities: a) skin from healthy fish; b) skin from stressed fish; c) skin from dead fish; d) from biofilm; e) from biofilters; f) from water.

Figure 4. Theta index based dendrogram of bacterial communities from different samples.

a) Samples from the control unit. Samples are represented by their sampling time (times 0 to 4). b) Samples from the test unit. Samples are represented by their sampling time (times 0 to 4). Sampling time: 1) before the stress trial (time 0), 2) stress trial at one week, 3) one week (time 1), 4) two weeks (time 2), and 5) three weeks (time 3), and 4 weeks after exposure to stress (time 4). Mucus bacterial communities from dead fish are also represented on this dendrogram.

Figure 5. Network of co-occurring genera based on correlation analysis.

A connection stands for a strong (Spearman’s r > 0.6) and significant (P-value < 0.01) correlation between two nodes (genera). The size of each node is proportional to the number of connections (i.e. degree). Colors indicate the phylum: red: Proteobacteria; blue: Actinobacteria; yellow: Firmicutes; green: Bacteroidetes.

Figure 6. Evolution of relative abundance of beneficial genus from skin fish mucus following a stress event.

Non-stress: sampling before stress; stress1: sampling 1 week after stress event; stress2: sampling 2 weeks after stress event; stress3: sampling 3 weeks after stress event; stress4: sampling 4 weeks after stress event. Statistical significant differences were tested by a Wilcoxon rank pair-test (p < 0.001).

Figure 7. Evolution of relative abundance of beneficial genus from skin fish mucus following a stress event.

Dead: sampling from Dead fish, Non-stress: sampling before stress; stress1: sampling 1 week after stress event; stress2: sampling 2 week after stress event; stress3: sampling 3 week after stress event; stress4: sampling 4 week after stress event. Statistical significant differences (represented by an asterisk) were tested by a Wilcoxon rank pair-test (p < 0.001).