Pinnipeds as sentinels of antimicrobial resistance

: mapping antimicrobial resistance genes in the waters of the United Kingdom

Abstract

Antimicrobial resistance (AMR) is a continual and growing threat to human and animal health. There is a lack of information about environmental reservoirs of AMR, many of which feed into the marine habitat. Pinnipeds are often used as sentinels of ocean health because they are long lived, apex predators that forage over wide areas and thus can represent the food web over large regions. They can also be relatively easily sampled when they are on land. Documenting the prevalence of AMR-containing microbes present in pinnipeds can thus provide a better understanding of the pathways of AMR dissemination. An assessment of AMR genes and bacteria in the environment can provide insight into the changing use of antimicrobials, but a formal assessment and synthesis of the current situation is lacking. This study aims to create a current baseline and characterization of the grey seal gut resistome and use associated telemetry data to evaluate possible sources and sinks of AMR in the waters surrounding the UK.
In Chapter Two, I used a meta-analysis and systematic review to examine the presence and prevalence of bacteria isolated from pinnipeds that possess AMR as reported in the literature. The protocol for this study is registered with the International Prospective Register of Systematic Reviews (PROSPERO) under registration number CRD42021220169. The initial search was refined to 35 studies for data extraction, done by three independent reviewers. All isolates reported belonged to species known to be pathogenic, and most studies did not focus on AMR, but reported AMR secondarily. This study found the most prominent resistance was to Lincomycin and Tetracycline antibiotics; many studies sampled more animals than they examined for AMR; it demonstrated there is an expanding need for standardized reporting system for AMR studies in pinnipeds; finally, it demonstrated the need to screen across different bacteria and at the molecular level to get a true idea of AMR prevalence.
In Chapter Three, I developed and tested a screen for AMR in seal faeces to characterize their gut resistome. I examined the most effective means to sample the faeces; the most effective way to extract the DNA; how DNA degrades over time in storage and through different extraction methods; and optimised and tested an end point PCR biased assay to determine the presence and absence of clinically and environmentally important AMR. This study determined that solid scats are preferred over the swab unless the sample will be in storage for some time, and that Phenol:Choloroform extractions are preferred over a commercially available kit. 361 individual genes were identified and extracted, with most resistance to Tetracycline, Beta-Lactam, and Aminoglycoside antibiotics. An average of 4.4568 different genes were found per faecal sample, and this study demonstrates that each animal carries a propensity for multiple resistance in their resistome, which could prove to be problematic in the future. This upholds the results of our meta-analysis in Chapter Two for Tetracycline but identifies two other significant classes – Beta-Lactam and Aminoglycoside antibiotics.
In Chapter Four, I determined the number of animals required to sample at minimum to find all positive genes of the genes in the designed screen using rarefaction curves. This Chapter also evaluated the relationships between animals’ resistomes and AMR gene classes. The null hypothesis was no significant relationship between location or animal and the resistome, and was tested using dendrograms, clustering, SIMPER, and Shannon diversity. The rarefaction curves suggested in excess of 30 animals is necessary to gain a population level view of the resistome. The resistome was significantly influenced by location and animal, suggesting there is an environmental influence on the AMR an animal carries. Resistance to Tetracycline, Aminoglycoside, and Sulfonamide antibiotics were significant in the models, meaning they were more represented than expected by chance, and three animals were determined to be significantly different from every other animal sampled. This upholds the results found in Chapter Two and Three, but identifies Sulfonamides, which were not identified in the initial literature search, as major contributors.
In Chapter Five, I used telemetry data to determine overlap between animal tracks, and whether there is an obviously discernable reason for the statistical differences determined in Chapters Three and Four. One complication of this study is the failure of telemetry tags, so not all animals run through the screen in Chapter Three could be tracked in this Chapter. Aquaculture, wastewater, and hospitals were mapped and there is a clear connection between AMR and anthropogenic influence, possibly from urban and industrial areas, because animals that used water by large centers of human population had resistomes that contained resistance to every antibiotic class tested. Areas that were statistically different in animal resistome in Chapter Four were mapped, and showed little spatial overlap with each other, strengthening the evidence that habitat influences resistome.
In conclusion, although Chapter Two reported the prevalence of AMR in isolates from pinnipeds at ~60%, this Thesis suggests overall AMR prevalence in the seal gut resistome is closer to 99%. This is likely because this uses molecular methods, rather than phenotypic screening. We have created a repeatable and standardized screen and examined a wide range of pinniped resistomes from different populations across the United Kingdom. To our knowledge this is the first systematic review of AMR in pinnipeds, and the first genetic study of latent resistance – or the possibility of resistance – in pinnipeds, rather than only on pathogens or bacteria of clinical importance. We can conclude that there is a statistical environmental influence on the resistome of pinnipeds, which may be environmental or anthropomorphic, and warrants further study to stop the spread of AMR in the waters surrounding the UK.

Date of Award	26 Jun 2025
Original language	English
Awarding Institution	Abertay University
Supervisor	Scott Cameron (Supervisor), Kimberley Bennett (Supervisor) & Debbie Russell (Supervisor)