High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments

Abstract

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Mākirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.

Population structure and pathogen interaction of Escherichia coli in freshwater: Implications of land‐use for water quality and public health in Aotearoa New Zealand

“…In parallel, the realization that E. coli may also persist in water, soil and sediment has provided new impetus to investigate the maintenance and survival of newly described environmental E. coli ‐like Escherichia spp. (Gilroy et al, 2021 ; Liu et al, 2015 ; van der Putten et al, 2021 ), isolated from terrestrial and aquatic habitats (Koh et al, 2022 ; Mire et al, 2022 ; Moinet et al, 2024 ). The advances in molecular methods to subtype E. coli and identify E. marmotae and E. ruysiae should allow new insights into their overall abundance and association with enteric pathogen species.…”

“…Overall, the non‐ E. coli Escherichia comprised only a minor proportion of the isolates subtyped using PCR (0.6%, 24/3980), with 23 E. marmotae and 1 E. ruysiae identified from 14 sites (Figure S1 ). Previous study from New Zealand has indicated that E. marmotae and E. ruysiae can be isolated from avian faeces, are present in wildlife and pristine bush habitats, and have fewer virulence factors compared to E. coli (Cookson et al, 2022 ; Moinet et al, 2024 ). Together with data from this study for example, where E. marmotae were negatively associated with the Ruminant MST marker (Table S6 ), these lines of information from recent studies, provide important evidence to water managers that despite being indistinguishable from FIB using standard culture‐based enumeration methods, the recently characterized Escherichia species are not associated with waterborne pathogens and represent a lower likelihood of potential health risk to recreational users of water.…”

“…Intriguingly, phylotype B2 has also been identified as a human‐associated E. coli phylotype, commonly isolated from urinary tract infections and associated with the extended‐spectrum beta‐lactamase resistant antibiotic resistance phenotype (Toombs‐Ruane et al, 2023 ). Therefore, we hypothesise that there are likely to be multiple pathotypes represented by phylotype B2, including those from forested sites (Petit et al, 2017 ), wildlife (Moinet et al, 2024 ) and humans with contrasting pathotype‐specific genetic characteristics (Johnson et al, 2017 ; Smati et al, 2015 ) which may influence their survival and persistence in the environment. The abundance of B2 in low impact sites less affected by faecal sources in this study contrasts with other studies where B2 were identified as lacking survival‐associated functions and were shown to survive for shorter periods in secondary environments, such as beach sand (Rumball et al, 2020 ; Rumball et al, 2023 ).…”

“…However, a risk assessment would be required to determine if pathogens are present at concentrations likely to affect human health. Analysis with additional MST markers such as cat, dog or possum ( Trichosurus vulpecula , a prevalent introduced pest) (Moinet et al, 2024 ) may provide clues on other faecal sources. The influence of sediments on microbial quality in the overlying water column has attracted increasing scrutiny with the proposal that sediments in water bodies can act as reservoirs for both FIB and pathogens (Devane et al, 2014 ; Devane et al, 2019 ; Petit et al, 2017 ).…”