Investigating wastewater pipe microbiomes in a large teaching hospital following endemic infection outbreaks

Culture-based approaches enabled the targeted isolation of extended-spectrum beta-lactamase (ESBL)-producing bacteria and other MDR pathogens, while metagenomics allowed for a comprehensive overview of the taxonomic and functional diversity of the microbiomes. Specific attention was paid to ARGs, including functional metagenomic screens to identify the phenotypic impact of these genes. Among these ARGs, a class D beta-lactamase, OXA-58 was amplified from a wastewater pipe.

This gene was cloned and expressed in a previous sensitive host, leading to a newly acquired resistant phenotype. This gene was also found to confer different phenotypic resistance to that suggested by its annotation, showing that phenotypic resistance is determined by more than just the presence of an ARG, but also by the host expression system.<br><br>The findings of this thesis revealed that hospital wastewater pipes harbour diverse microbial communities, including numerous clinically significant MDR organisms such as ESBL-producing Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Elizabethkingia meningoseptica.

These pathogens demonstrated resistance to a wide range of antibiotics, including carbapenems, highlighting the role of wastewater systems in fostering and disseminating ARGs. Functional analyses revealed the activity of key ARGs, including beta-lactamases, and their contribution to resistance phenotypes. Clinical isolates obtained from patients with HAIs were also examined, providing critical insights into the MDR profiles and AMR mechanisms within these strains.

All clinical isolates demonstrated significant phenotypic resistance to commonly used antibiotics, and harboured ARGs associated with resistance to beta-lactams, aminoglycosides, and fluoroquinolones, among others. Notably, a novel species, Pseudocitrobacter limerickensis, was identified and characterised, which revealed its MDR nature. This is of particular concern given its ability to colonise humans in clinical settings. <br><br>Biofilm formation and eradication was a significant focus of this study, as biofilms are known to enhance the persistence and resilience of bacteria in wastewater environments.

Many isolates demonstrated robust biofilm-forming capabilities, as evaluated by crystal violet assays. Biofilm formation varied across bacterial species and pipe sections, with isolates from certain pipe sections consistently exhibiting stronger biomass formation. These findings suggested environmental factors, such as pipe geometry and water flow dynamics, may influence the selection of biofilm-forming bacteria.

The presence of ARGs within biofilms further emphasises their role as reservoirs for MDR pathogens and resistance genes, posing risks to both environmental microbiota and hospital infection control. The efficacy of commonly used disinfectants, namely sodium hypochlorite, OptizanTM, VirkonTM, and ClinellTM, was investigated against biofilm-forming, ESBL-producing bacteria isolated from hospital wastewater pipes. Commonly used hospital disinfectants demonstrated efficacy against planktonic bacteria.

They were also capable of eradicating biofilm effectively, although to a more limited degree compared to killing planktonic cells. Different isolates exhibited varied survival ability against disinfectants, even within the same species, suggesting prediction of disinfectant susceptibility based on the causative organism at species level would not be accurate. This highlights the importance of monitoring and managing hospital wastewater systems as part of broader infection prevention and control strategies.

The findings also demonstrate the need for effective disinfection protocols and infrastructure design improvements to mitigate the risks posed by wastewater systems.<br><br>This thesis provides a comprehensive analysis of the role of hospital wastewater systems in harbouring and disseminating MDR pathogens and ARGs, contributing to the growing body of evidence linking environmental reservoirs to clinical infections. By combining advanced molecular techniques with traditional microbiological methods, this study offers valuable insights into the microbiome dynamics, resistance mechanisms, and biofilm formation within wastewater pipes.

The findings underscore the urgent need for targeted interventions to address the risks associated with wastewater systems in healthcare settings. Recommendations include the implementation of specialised disinfection protocols, infrastructure redesign to minimise biofilm formation, and enhanced surveillance of wastewater microbiomes to identify emerging threats.<br>