Comparison of antimicrobial resistant Enterobacterales isolates from the dairy production environment in low and high zinc containing regions.

Heavy metals occur naturally in the environment, and their concentration varies in soil across different regions. However, the presence of heavy metals may influence the antimicrobial resistance (AMR) in bacterial populations. Therefore, the objective of this study was to investigate and characterise the antimicrobial resistance profiles of Enterobacterales in soil and bovine milk filters from high and low zinc-containing regions in Ireland. In total, 50 soil samples and 29 milk filters were collected from two geographic locations with varying soil zinc concentrations. Samples were cultured for the enumeration and detection of Enterobacterales. Specifically, extended-spectrum beta-lactamase-producing Enterobacterales, carbapenem-resistant Enterobacterales and ciprofloxacin-resistant Enterobacterales were isolated using selective media. Species identification was performed using MALDI-TOF. The phenotypic resistance profiles of selected Enterobacterales were determined by disk diffusion testing, following EUCAST and CLSI criteria; while, the genotypic resistance profiles of the same isolates were determined by whole genome sequencing (WGS). Heavy metal concentrations were also measured for all soil samples. A total of 40 antimicrobial resistant Enterobacterales were identified in soil (n = 31) and milk filters (n = 9). The predominant species detected in the high zinc-containing region was Escherichia coli in both sample types (soil n = 10, milk filters n = 2), while in the low zinc-containing region Serratia fonticola was predominant in soil samples (n = 8) and E. coli in milk filters (n = 4). Ten E. coli isolates identified from soil samples in the high zinc-containing region were multidrug resistant, showing resistance to all the antimicrobials tested, except for carbapenems. The WGS findings confirmed the phenotypic resistance results. Moreover, zinc resistance-associated genes and genes encoding for efflux pumps were identified. The current study revealed distinct phenotypic resistance profiles of Enterobacterales in low and high zinc-containing regions, and highlighted the benefit of utilising milk filters for AMR surveillance in dairy production.

Characterization of antimicrobial resistant Enterobacterales isolated from spinach and soil following zinc amendment.

Antimicrobial resistant bacteria can occur in the primary food production environment. The emergence and dissemination of antimicrobial resistance (AMR) in the environment can be influenced by several factors, including the presence of heavy metals. The aim of this study was to examine the presence and characteristics of antimicrobial resistant Enterobacterales in soils and spinach grown in soils with and without zinc amendment. A total of 160 samples (92 soil and 68 spinach) were collected from two locations, in which some plots had been amended with zinc. Samples were cultured on selective agars for detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL), carbapenem-resistant Enterobacterales and ciprofloxacin-resistant Enterobacterales. Samples were also cultured for enumeration of total Enterobacterales. Isolates were identified by MALDI-TOF. Antimicrobial susceptibility testing was carried out in accordance with EUCAST and CLSI criteria. The whole genome sequence (WGS) of selected isolates was determined. Inductively coupled plasma atomic emission spectrometry was also performed on soil samples in order to measure the concentration of zinc. In total 20 antimicrobial resistant Enterobacterales were isolated from the soil (n = 8) and spinach samples (n = 12). In both sample types, Serratia fonticola (n = 16) was the dominant species, followed by Escherichia coli (n = 1), Citrobacter freundii (n = 1) and Morganella morganii (n = 1) detected in spinach samples, and Enterobacter cloacae (n = 1) detected in a soil sample. The WGS identified genes conferring resistance to different antimicrobials in agreement with the phenotypic results; 14 S. fonticola isolates were confirmed as ESBL producers and harboured the blaFONA gene. Genes that encoded for zinc resistance and multidrug efflux pumps, transporters that can target both antimicrobials and heavy metals, were also identified. Overall, the findings of this study suggest the presence of zinc did not influence the AMR Enterobacterales in soil or spinach samples.