Antibiotic resistance represents a serious threat to human health and a relevant environmental contaminant. Antibiotic-resistant bacteria (ARB) and harboured antibiotic resistance genes (ARGs) were described in different settings, mainly in clinical contexts, but also in wastewater treatment plants or agricultural soil. In the environment, the general pollution context may favour the persistence or proliferation of ARB due to multiple genetic characteristics. Monitoring of ARGs and ARB in the environment plays an important role in unveiling sources and paths of dissemination. To address these issues this thesis explored three topics based on antibiotic resistant Pseudomonas aeruginosa and on soil: i) the biases that may be imposed by the high limits of quantification of ARGs in soil; ii) the survival of an exogenous ubiquitous bacterial strain (blaVIM+ P. aeruginosa) in soil and the possible effects of metal salts; and iii) the inferred interplay between phylogeny and accessory genome in distinct genotypes of carbapenem resistance (blaVIM+ or blaNDM+ P. aeruginosa). The first work (chapter 3) aimed to assess the limit of quantification (LOQ) for ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) in soil, based on the hypothesis that low doses of ARB and ARGs in soil are not quantifiable with current qPCR techniques, mainly due to DNA extraction procedures. To determine the LOQ, microcosms (10 g of agricultural soil, potting soil, sand, fallow soil and compost) were spiked with wastewater isolated ARB doses ranging from 102 to 107 CFU/g of dry soil. These spiked ARB harboured respectively the vancomycin resistance gene vanA (E. faecalis), quinolone resistance gene qnrS (Escherichia coli), and β-lactam resistance genes blaTEM (E. coli), blaOXA (E. coli, Acinetobacter johnsonii), blaIMP (A. johnsonii), blaVIM (P. aeruginosa) based on which the LOQs were determined. The microcosms were sampled to enumerate bacterial colony forming units (CFU), and extract DNA for ARGs quantification by qPCR. The LOQ was determined to be 104 copies of ARG per g of dry soil, independently of the soil type. Below this limit, it was not possible to quantify ARGs even when the respective host ARB could be cultivated. The results support the hypothesis that LOQ values are relatively high and may suggest the absence of ARGs in situations in which these may represent a threat. The second work (chapter 4) aimed at assessing the metal impact on the survival of a hospital effluent blaVIM+ P. aeruginosa and on the soil microbial community’s diversity. Microcosms were prepared with agricultural soil non-amended or amended with copper and zinc sulfate or nitrate aged for one month, and spiked with known doses of blaVIM+ P. aeruginosa. The ARB survival was monitored based on CFU enumeration and quantification of selected genes - extracytoplasmic function sigma factor, ecf, Verona Integron–encoded Metallo-β-Lactamase, blaVIM, class 1 integron- integrase gene, intl1, over 30 days. In addition, the microbial community composition (V3-V4 16S rRNA gene amplicon sequencing) was analysed. Over this period, the P. aeruginosa content (CFUs/g dry soil) and ecf and blaVIM (copy number/g dry soil) decreased in all the tested conditions but was still quantifiable after 30 days. This confirms the ARB persistence in soil along the 30 days, excluding the hypothesis of ARG loss during this period. Microbiome analysis revealed a clear influence of metals in the bacterial community diversity, independent of the metal type and salts nature. This study permitted to conclude that the metal amendment affects the soil microbial quality but has a negligible impact on the exogenous bacteria survival. These results highlight the importance of considering microbial interaction and characteristics, such as metal tolerance, in the assessment of ARB persistence in the environment. In chapter 5 the genomes of blaVIM-2+ or blaNDM-1+ P. aeruginosa strains were compared. The ARG blaVIM-2 is mostly observed in Pseudomonas species, while blaNDM-1 is distributed among distinct genera and orders. The work focused on phylogenetic distribution and genomic features of a dataset of 116 blaVIM-2+ and 27 blaNDM-1+ genomes, from 38 countries. The selected genomes were annotated and the core sequence multilocus sequence typing (MLST) were determined. The blaVIM-2+ and blaNDM-1+ genomes were analysed using a comparative genome approach to assess the core and accessory genes, later used to determine the bacteria antibiotic resistance and functional profile. To describe the blaVIM-2 and blaNDM-1 genomic environment, the flanking regions were annotated through sequence comparison. The phylogenetic and geographic distribution analyses suggested a worldwide distribution of the strains belonging to several STs with cases of endemism. The blaVIM-2 + and blaNDM-1 + accessory genomes presented different antibiotic resistance and functional profiles, regardless the majority of the ARGs and proteins families were shared. Interestingly, the copresence of blaVIM-2 and blaNDM-1 and other carbapenems resistance genes in different genomes was observed. The genomic environments of the two ARGs were different, being blaVIM-2 associated with distinct transposons structures (Tn21, Tn402-like mostly) and blaNDM-1 to different elements (ISAba125 and bleMBL and IS91). This work emphasized the importance of considering different approaches to tackle the spread of carbapenem resistant bacteria evaluating the phylogeny, and geographical distribution but mostly the genomic characteristics of the strains. Our works aimed to indicate possible weaknesses to be improved in antibiotic resistance monitoring and highlight the ARB phylogenetic role and genetic characteristics favouring the ARGs spread. In particular, the experimental work evidences the possible survival of ARB in soil, mostly in extremely polluted conditions. Moreover, the survival and presence of these ARB in soil could avoid the quantification by molecular biology methods due to their high LOQ. The study of the ARB genomic characteristics may be useful to prevent the adaptation to environments and to find additional biomarkers for their monitoring.
Date of Award | 10 Feb 2023 |
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Original language | English |
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Awarding Institution | - Universidade Católica Portuguesa
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Supervisor | Célia Manaia (Supervisor), Olga Cristina Pastor Nunes (Co-Supervisor) & Ivone Vaz-Moreira (Co-Supervisor) |
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