Characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators

Abstract

Background: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring.

Methodology/principal findings: Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and alpha-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies.

Conclusions/significance: This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.

Figure 1. Sampling sites along Mission (M4–M9) and Laguna Channel (M2 and M3) watersheds.

Samples were delineated into different habitat types: creek (M3, M5–M9, where M6 and M9 were from drains), lagoon (M2 and M4), and ocean (M1). Open circles (○) represent storm drains, and filled circles (•) represent creek, lagoon or ocean sites.

Figure 2. NMDS plot of PhyloChip community distances.

Bray-Curtis metric was used, and a stress of 8.14 was obtained. Each site is represented by a different color. The grey lines delineate grouping of creek, lagoon, ocean and fecal samples.

Figure 3. Bacterial community composition comparison across fecal, lagoon, creek and ocean samples.

(A) Distribution of relative richness at the class level. Number of OTUs in each sample types were divided by the total count for each sample type as indicated in parentheses on the x-axis. (B) Relative richness of Bacilli, Bacteroidetes and Clostridia detected. (C) Relative richness of α-proteobacteria detected. (D) Bacillus, Bacteroidetes, Clostridia to α-proteobacteria ratios (BBC∶A).

Figure 4. Boxplots of UniFrac similarity metrics between water and fecal-sample-associated OTUs (FSAO).

(A) Variable subpopulations. (B) Stable populations. Each box represents similarity metrics from all 3 days at each site. Boxplots with different letters indicate significant differences (p-value<0.05), compared using the student t-test. The samples were arranged from upstream to downstream (left to right) for samples M9-M4, and M3-M2.

Figure 5. Bacillus, Bacteroidetes, Clostridia to α-proteobacteria ratios (BBC∶A) from each site.

Ratio from each day is represented by a bar of different color.

Figure 6. Bacillus, Bacteroidetes, Clostridia to α-proteobacteria ratios (BBC∶A) of communities analyzed by sequencing or PhyloChip.

Sample types include, gut (•), sewage-associated (▪), and non-fecal (♦) associated samples. Unfilled diamond symbols (⋄) represent non-fecal samples from anoxic environments.