Transfer of antibiotic resistances between enterococci in biofilm

Abstract

Antibiotic resistance is an urgent and increasing problem in human and animal healthcare. While it is recognised that the environment must provide an opportunity for resistance to develop and spread, direct evidence for the mechanisms involved is still lacking. This research utilised enterococci from an agrarian environment to demonstrate horizontal gene transfer (HGT) of antibiotic resistance genes in specific reservoirs.

Screening a biobank of over 600 environmental isolates referencing previous partial characterisation data resulted in the selection of eleven Enterococcus faecalis and four E. faecium strains that exhibited potential for conjugation via a pheromone-dependent pathway. These isolates had prolific and diverse antimicrobial resistance profiles. Conjugal transfer of antibiotic resistance phenotypes was determined using a solid agar mating method followed by a standard antibiotic selection test resulting in different transfer patterns. Multiple gene transfer was observed in single reactions. An interspecies conjugal transfer of vancomycin resistance from E. faecalis to E. faecium was identified while the remaining reactions were within the same species. Transfer efficiencies ranging from 2 × 10−1 to 2.3 × 10−5 were determined. Interspecies transfer of vancomycin resistance among environmental isolates of enterococci had not previously been characterised, along with alternating transfer of different determinants from the same donor to different recipients. In certain cases, antimicrobial resistance to non-transferred resistance was elevated in transconjugants (T1, T2 and T4).

A novel biofilm apparatus model, based upon a Gene Frame, was developed to allow nondestructive analysis of Enterococcus biofilm. Fluorescently tagged Concanavalin A was used to label extracellular matrix material and bacteria were identified by fluorescent in situ hybridization (FISH) and DAPI staining. This unique model was more reproducible X than standard biofilm assays and it proved to be flexible in that it was adapted to identify antibiotic resistance genes. This novel system was used to demonstrate that interspecies transfer of vancomycin resistance takes place in bacterial biofilms, which are considered to be the natural state for environmental bacteria. Additionally, multiprobe FISH targeted to vanA on mobile elements demonstrated for the first-time vancomycin staining inside enterococcal biofilm.

Another potential reservoir of bacteria in an aquatic environment is the freshwater sponge. Enterococcus conjugation experiments were performed on Ephydatia fluviatilis and Spongilla lacustris, two sponge species that exist in similar geographical topography to where the enterococci were isolated. Enterococci were shown to bind to sponge material and HGT of vancomycin resistance was demonstrated in both sponge species by the modified FISH assay and by direct antibiotic selection methods.

Overall, this thesis highlights that enterococci of environmental origin are capable of transferring important resistance determinants in and out of biofilm, of their own construction. Their survival under harsh environmental conditions, such as low temperature and nutrient limitation, reduced but did not eliminate their ability to conjugate. Enterococcus faecalis and faecium have the potential to propagate antimicrobial resistance in the natural environment. An assessment of the impact of environmental conditions on HGT rates could help to preserve useful antibiotics from selection for resistance

Date of Award	Feb 2018
Original language	English
Sponsors	Department of Education and Learning (DEL)
Supervisor	James Dooley (Supervisor) & Patrick Naughton (Supervisor)