Resistance of environmental bacteria to heavy metals and antibiotics in selected New Zealand soils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand

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The usage of superphosphate fertilizers, animal remedies and other material containing heavy metals (HMs) in agriculture and horticulture is a problematic issue resulting in the accumulation of HMs in the soil. The presence of these HMs in the soil leads to the induction of resistance of environmental bacteria to these heavy metals and may co-select for resistance against a broad range of antibiotics (Abs). This co-selection may increase the health risk for both humans and livestock because of resistance to the wide range of Abs. As well as direct health effects, an increase in Ab resistance may impose a significant burden on the livestock industry and primary production, leading to potential for immense social and financial losses. The current project was aimed to investigate the resistance of soil-borne bacteria sampled from selected regions of New Zealand. Genetic diversity of these bacteria and molecular aspects of horizontal transfer of HM and Ab resistance genes to other bacterial hosts was also investigated. Soil samples with a different history of usage, including pastoral and arable with high levels of HMs (e.g. cadmium (Cd) and zinc (Zn)) were collected from the Waikato region (WR), as well as soil from an area of native bush (background) as control. Waikato Region (WR) is one of the regions in New Zealand with high levels of HMs in soil, due to the regular use of HM-containing superphosphate fertilizers and animal remedies. Belmont Regional Park (BRP) airstrip soil was used as a novel site to explore bacterial communities’ resistance to HMs, and any co-selected Ab resistance. A comprehensive investigation was performed to simulate the soil environment contaminated with various levels of HMs to interrogate induced resistance to HMs and Abs in soil bacterial communities using microcosms with 6 weeks and 6 months incubation. The experiments carried out to investigate soil bacterial resistance to HMs and Abs were divided into two different categories, including physiological and molecular experiments. The physiological tests included plate culturing with a range of HMs and Abs concentrations and Pollution Induced Community Tolerance (PICT) analysis. Molecular investigations using Terminal Restricted Fragments Lengths Polymorphism (TRFLP) and Next Generation 16s rDNA were conducted to determine the probable changes in bacterial community structures induced by selection pressure of HMs presence in soil samples. Cd resistance genes were detected in individual bacterial isolates using specific oligomeric DNA primers via the polymerase chain reaction (PCR). Horizontal transfer of these genes to new bacterial recipients was investigated. Finally, Cd resistant bacterial isolates involved in Horizontal Gene Transfer (HGT) were identified using 16s rDNA Sanger Sequencing. Results clearly showed that there were significant differences between the levels of resistance to HMs and Abs in bacterial isolates from WR’s pastoral and arable soils compared to background soil (native bush). Differences between BRP soil samples with higher levels of HMs compared to those with lower HMs concentrations, and also microcosms’ with a range of HM levels showed there were significantly greater number of bacterial isolates resistant to HMs and Abs in soils with the higher initial levels of HMs. Pollution Induced Community Tolerance (PICT) analysis provided complementary results in concordance with the results of plate culturing experiments and showed the higher levels of bacterial resistance to HMs and Abs in soils with the higher initial levels of HMs. Terminal Restriction Fragment Length Polymorphism (TRFLP) and 16s rDNA Next Generation Sequencing experiments investigated HM-induced bacterial communities' structure changes and revealed significant differences among the bacterial community structures in the selected BRP and microcosms soil samples. The HGT experiments revealed the horizontal transfer of Cd resistance genes from donor isolates (from WR, BRP, and microcosms soils) to a characterised recipient bacterial strain in vitro, suggesting these genes were carried by mobile genetic elements. Overall, the result of the current project showed that there were higher levels of bacterial resistance to HM and also to Ab occurred while different levels of HMs were present in the soil. In addition, higher levels of HM and Ab resistance induction occurred in the presence of specific concentrations of HMs in microcosms’ soils. The bacterial community structures were changed in the presence of various levels of HM in soil. The investigation of bacterial community structures changes in microcosms containing background soil samples were greater compared to the microcosms containing pastoral soils; it is concluded in higher changes in bacterial communities in soils in presence of selection pressure of HMs. Cd resistance genes located on mobile genetic elements were able to be transferred horizontally form donor bacterial strains to recipients and the transconjugants showed resistance not only to Cd, Zn and/or Hg, but also to a range of Abs; it showed the possibility of spread of these HM resistance genes to the new bacteria in soil and conferring HM and subsequent Ab resistance in recipients.
Figures 2.2 (=Soucy et al., 2015 Fig 1a-f) & 2.3 (=Noar & Gophna, 2013 Fig 1) have been removed for copyright reasons.
Soil microbiology, New Zealand, Heavy metals, Environmental aspects, Drug resistance in microorganisms, Bacterial genetics