AbstractThis thesis aimed to establish the potential of extremely halophilic microorganisms, isolated from Kilroot Salt Mine, as a reservoir of unexplored natural product chemistry with antimicrobial activity against the clinically relevant ESKAPE pathogens. The capacity of haloarchaea to modulate bacterial quorum sensing via the production of cross-domain induction and inhibitory compounds were also investigated.
Both conventional and enhanced culturing methods including the iChip device were employed to obtain a collection of morphological/genetically distinct halophilic bacteria and archaea from brine samples and PFA, resulting in a collection of 51 Haloarchaea and 10 halotolerant Actinobacteria isolates.
Crude extracts from these haloarchaea and bacteria isolates, alongside 16 extremophilic fungi were investigated for antimicrobial activity against the ESAKPE pathogens and QSI production. The screening revealed extracts from Brevibacterium sp., NAT-30 and NAT-27, exhibited antimicrobial against MRSA at 2 mg/mL and 0.5 mg/mL respectively. The isolate Halorubrum sp. iNAT-14 exhibited antimicrobial activity against P. aeruginosa and A. baumannii, with a MIC of 4 mg/mL. The most potent haloarchaea extract was from Haloferax volcanii DS2 with a MIC of 0.5 mg/mL against E. faecium. The yeast-like fungus, A. pullulans EXF-9635, displayed the most potent antimicrobial activity at 0.125 mg/mL against MRSA.
Furthermore, this extract exhibited anti-biofilm activity with a MBIC of 2 mg/mL, and 1 mg/mL against MRSA and Staphylococcus aureus biofilms, respectively. In vitro studies revealed no toxicity at concentrations ≤ 2 mg/mL using NCTC 929 cell line and in vivo analysis with G. mellonella larvae revealed 80% survival in concentrations ≤1 mg/larvae. The highest percentage survival of infected larvae was at 0.125 mg/larvae.
The extract of Halorubrum sp. CSM52 was demonstrated to induce bacterial QS using the bioreporters A. tumefaciens NTL4(pZLR4), E. coli pSB401, pyoverdine production in PAO-MW1, and pyocyanin inhibition in PAO1. Initial characterisation of the bioactive(s) using TLC-overlays, biochemical tests, and LC-MS/MS analysis excluded structural similarity to known AHL molecules, suggesting that it may be a chemically modified AHL or a DKP. Fractionation of the crude extract revealed that fraction nine induced pyoverdine production, while fraction 4 and 6 were capable of inhibiting pyocyanin production in PAO1. Initial results suggest the role of this AI may be involved in haloarchaeal biofilm formation.
Metagenomic analysis of Kilroot Salt Mine using Kaiju and Kraken2 classifiers, revealed a reduction in the relative abundance of Salinobacter spp. and Haloquatrum spp. compared to hypersaline lakes. Metagenome mining revealed both bacteria and archaea harbour AMR genes in a relatively undisturbed environment. Genomic mining of brine and salticle metagenomes for halocin AMPs revealed that several halocin-S8 homologs existed. A putative AMP termed, Halocin-KSM was developed from a consensus sequence of known halocin homologs and was predicted to possess antimicrobial activity using in silico analysis. This putative activity was verified using disc diffusion and MIC assays, revealing that the C-terminal amide species of Halocin-KSM had a MIC of 0.06125 mg/mL against S. aureus.
Both culture-dependent and culture-independent approaches have provided a detailed insight into the microbiome of Kilroot Salt Mine and provide an excellent framework for optimising future isolation procedures. Structural elucidation of bioactives from Halorubrum sp. CSM52, A. pullulans EXF-9635, and Haloferax volcanii DS2 will provide an invaluable insight mechanism by which halophilic microbes exert their activity. In conclusion, these results have established that halophilic microorganism from all three domains of life are capable of producing antimicrobial compounds against clinically relevant pathogens.
|Date of Award||Dec 2020|
|Sponsors||Northern Ireland Department for the Economy|
|Supervisor||Brendan Gilmore (Supervisor), John McGrath (Supervisor) & Panagiotis Manesiotis (Supervisor)|
- antimicrobial resistance
- quorum sensing