Investigating the role of heavy metals in the environment as a selective pressure for the dissemination of antimicrobial resistance

Antimicrobial resistance (AMR) is a growing concern globally. The ability of microorganisms to survive in the presence of antimicrobials and the spread of antimicrobial resistance genes (ARGs) pose a significant risk to human, animal and environmental health. The environment's impact on the emergence and spread of AMR is increasingly acknowledged as critical, with reservoirs like soil and water playing a key role in the persistence and dissemination of antimicrobial-resistant bacteria (ARB) and ARGs. In this regard, the primary food production environment is of considerable importance due to the significant potential of ARB and ARGs to be transmitted to humans and animals through the food chain. Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem resistant Enterobacterales (CRE) have been recognised by the World Health Organization as critical priority pathogens due to the severe infections they can cause, which are difficult to treat and often require last-resort treatments. Therefore, the surveillance of these bacteria in the environment is fundamental to tackle AMR. Beyond antimicrobials, also other factors, such as heavy metals and biocides, influence AMR. Several mechanisms underlie the association between heavy metals and AMR, including co-resistance, cross-resistance, co-regulation and biofilm formation. Therefore, the aim of this research was to investigate the role of heavy metals, in particular zinc, as a selective pressure for antimicrobial resistant Enterobacterales’ occurrence and dissemination in the primary food production environment. Firstly, a scoping review was conducted to identify the knowledge gaps in this area (Chapter Two). A total of 73 studies undertaken over a period from 2008 and 2021 were included, the majority of which were conducted in China. The main environmental samples analysed in these studies were soil, water and manure, collected from different settings, including areas with natural occurrence of heavy metals, areas intentionally amended with heavy metals, and areas close to mines or industries that might release heavy metals. The results underlined the link between heavy metals and AMR, with particular focus on the role of mobile genetic elements (MGEs) in the dissemination of ARGs and heavy metal resistance genes (MRGs). Following this, a research study was performed to assess the presence and characteristics of antimicrobial resistant Enterobacterales in soil and spinach samples with and without zinc soil amendment (Chapter Three). A total of 160 samples (soil, n = 92, spinach, n = 68) were collected from two different locations, where some of the soil plots were amended with zinc sulphate and other plots were used as control. A total of 20 antimicrobial resistant Enterobacterales were isolated, with Serratia fonticola species being the most common (n=16). None of the isolates were resistant to three or more antimicrobials. Genes encoding antimicrobial resistance and genes encoding zinc resistance were identified. However, a direct correlation between zinc amendment of soils and AMR was not identified. A follow on research study was conducted to evaluate the occurrence of antimicrobial resistant Enterobacterales in dairy production (Chapter Four). Fifty soil samples and 29 bovine milk filters were collected from ten different farms (five farms from two different geographical regions in Ireland with low and high zinc concentrations). A total of 40 antimicrobial resistant Enterobacterales were identified, with Escherichia coli the most predominant species. Phenotypic analysis identified 17 multidrug resistant (MDR) isolates, including ten E. coli isolates collected from the high zinc containing region, and seven MDR out of nine isolates from milk filters. Genotypic analysis identified ARGs and MRGs among the isolates collected from the high zinc containing region; however, no direct correlation between zinc resistance genes and AMR was observed. The advantage of using milk filters for AMR monitoring in dairy production, which allows early detection of ARB before entering the food chain, was recognised. Finally, a metagenomic study on ten composite soil samples and 18 milk filters, collected as part of the research detailed in Chapter Four, was performed to acquire a better understanding of the AMR presence in dairy production in high and low zinc containing regions (Chapter Five). This study identified the dominant microbial communities in both sample types, as well as the diverse profile of ARGs and MRGs present in the different sample types. Overall, this body of work demonstrates that the primary food production environment can harbour clinically relevant Enterobacterales, serving as a significant reservoir and transmission source of AMR. This highlights the critical need of monitoring strategies to address the spread of AMR within the food chain, to which humans and animals may be exposed, and protect public health. Milk filters provide an ideal opportunity for the surveillance of AMR in dairy production. Although a direct correlation between zinc and AMR occurrence was not observed in the studies undertaken, the potential role of other heavy metals, such as copper, was suggested. Further investigation is needed to understand the impact of other heavy metals on AMR dynamics in the primary food production environment.

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University of Galway

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University of Galway Theses (PhD Theses)