Transcriptomic and rRNA:rDNA Signatures of Environmental versus Enteric Enterococcus faecalis Isolates under Oligotrophic Freshwater Conditions

Abstract

The use of enterococci as a fecal indicator bacterial group for public health risk assessment has been brought into question by recent studies showing that "naturalized" populations of Enterococcus faecalis exist in the extraenteric environment. The extent to which these naturalized E. faecalis organisms can confound water quality monitoring is unclear. To determine if strains isolated from different habitats display different survival strategies and responses, we compared the decay patterns of three E. faecalis isolates from the natural environment (environmental strains) against three human gut isolates (enteric strains) in laboratory mesocosms that simulate an oligotrophic, aerobic freshwater environment. Our results showed similar overall decay rates between enteric and environmental isolates based on viable plate and quantitative PCR (qPCR) counts. However, the enteric isolates exhibited a spike in copy number ratios of 16S rRNA gene transcripts to 16S rRNA gene DNA copies (rRNA:rDNA ratios) between days 1 and 3 of the mesocosm incubations that was not observed in environmental isolates, which could indicate a different stress response. Nevertheless, there was no strong evidence of differential gene expression between environmental and enteric isolates related to habitat adaptation in the accompanying mesocosm metatranscriptomes. Overall, our results provide novel information on how rRNA levels may vary over different growth conditions (e.g., standard lab versus oligotrophic) for this important indicator bacteria. We also observed some evidence for habitat adaptation in E. faecalis; however, this adaptation may not be substantial or consistent enough for integration in water quality monitoring. IMPORTANCE Enterococci are commonly used worldwide to monitor environmental fecal contamination and public health risk for waterborne diseases. However, closely related enterococci strains adapted to living in the extraenteric environment may represent a lower public health risk and confound water quality estimates. We developed an rRNA:rDNA viability assay for E. faecalis (a predominant species within this fecal group) and tested it against both enteric and environmental isolates in freshwater mesocosms to assess whether this approach can serve as a more sensitive water quality monitoring tool. We were unable to reliably distinguish the different isolate types using this assay under the conditions tested; thus, environmental strains should continue to be counted during routine water monitoring. However, this assay could be useful for distinguishing more recent (i.e., higher-risk) fecal pollution because rRNA levels significantly decreased after 1 week in all isolates.

Keywords: bioinformatics; environmental microbiology; fecal organisms; metatranscriptomics; public health; rRNA; water quality.

FIG 1

Comparison of changes in viable cell counts (A) and rRNA:rDNA ratios (B) of enteric versus environmental E. faecalis isolates over time in dialysis bag mesocosms. Three enteric and three environmental isolates are represented by different shades of orange and blue, respectively. Error bars are standard deviations among three technical replicates.

FIG 2

Differentially expressed genes between enteric and environmental E. faecalis isolates between days 1 and 3. Shown are functional genes that were significantly more expressed (P_adj < 0.05) in enteric relative to the environmental isolates (negative log₂ fold difference) or significantly more expressed in environmental relative to enteric isolates (positive log₂ fold difference). Error bars represent the standard errors among biological replicates. None of the putative, habitat-specific auxiliary genes were significantly differentially abundant, and thus, they are not shown here.

FIG 3

Cellular abundance and rRNA:rDNA ratios for E. faecalis MTUP9 (an environmental isolate) under standard pure culture conditions. The same strain was grown in three separate batch cultures (biological replicates) and at each time point, each biological replicate was sampled 3 times (technical replicates) for nine total measurements at each time point. These nine measurements were averaged, and error bars are standard deviations of biological and technical replicates.