Antibiotic resistant bacteria and antibiotic resistance genes are considered contaminants of emerging concern, nowadays widely disseminated in the environment. Urban wastewater treatment plants are major recipients and reservoirs for these contaminants. In urban these plants, wastewater is subjected to different types of treatment that reduce the levels of antibiotic resistant bacteria and antibiotic resistance genes, although not completely. Considering that most of the bacteria are not culturable and that this fraction might harbor antibiotic resistance genes, culture-independent methods are currently used to assess antibiotic resistance in the environment. Among these methods, quantitative PCR is considered the gold standard used to quantify antibiotic resistance genes in environmental samples, although the lack of harmonized methods seriously limits the reliable comparison of results obtained in different laboratories. Among the antibiotic resistant bacteria emitted by wastewater treatment plants, Enterobacteriaceae represent an important fraction and among these Klebsiella pneumoniae deserve special attention. Indeed, K. pneumoniae is a bacterial species that besides the clinical importance when associated to humans, can also be found in the environment, in soils, plants, water, and wastewater. The capacity of this bacterial species to thrive in different environments and in humans and animals increases its significance in terms of human health threat might constitute a human health threat. However, the traits that might be maintained or lost during the transit of K. pneumoniae through clinical and environmental contexts are still unknown. This thesis aimed to 1) advance the knowledge regarding the use of harmonized analytical quantitative PCR methods that might enable reliable comparisons of genes quantification ; 2) design a cell-based internal standard that could be used in different laboratories to assess losses during water samples filtration, DNA extraction and quantitative PCR quantification; and 3) contribute to a better understanding of the ecology of K. pneumoniae and infer about possible dynamics between clinic and environmental niches, with special focus on genetic diversity and antibiotic resistance stability. To tackle the first aim, genetic determinants encoding resistance to sulfonamides (sul1 and sul2), quinolones (qnrS), and β-lactams (blaTEM) and the 16S rRNA gene were monitored in DNA extracts supplied by partners who were investigating wastewater treatment processes, at full- or pilot-scale. In parallel, the influence on genes quantification of DNA shipment, quantitative PCR protocols, standards and equipment was studied in an interlaboratory comparison. These results and the literature available justified the efforts to meet the second aim. An internal standard, consisting in a cloned gene fragment not found in wastewater samples was designed and tested. This internal standard is to be used to spike wastewater or water samples aiming to control DNA losses during the processing of the sample and DNA extraction process. The emission of antibiotic resistant bacteria by wastewater treatment plants is an issue of concern, however it is not clear if these bacteria will survive and maintain their features once in the environment. To investigate this topic, K. pneumoniae was used as a model species and two distinct research approaches were used. A group of 3 rd generation cephalosporin-resistant K. pneumoniae isolates (25 wastewater; 34 clinical) was compared based on phenotypic, genotypic and genomic analyses (n=22) and a broader group of genomes collected from a public database (21 countries, 61 environmental; 78 clinical) was compared based on a core and pangenome approach and profiles of antibiotic and metal resistance, virulence, efflux systems, oxidative stress and quorum sensing traits. According to the results obtained and their analysis, it was concluded that wastewater treatment efficiency and wastewater quality regarding antibiotic resistance emissions should always be measured based on absolute abundance (per volume), rather than in relative abundance (per total bacteria). The interlaboratory comparison seemed to be reliable, although DNA extract quality and stability during shipment, as well as consumables and equipment specificities, may be critical for the monitoring findings. The use of a cell-based internal standard may contribute to overcome those limitations. This internal standard permitted to estimate the water matrix effect which was associated with an underestimation that ranged 0.1–0.9 log gene copy number mL−1 of sample, irrespective of the water type. Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization, although distinct lineages may prevail in clinical or environmental settings. Genetic determinants related to efflux, oxidative stress or quorum sensing functions were common to clinical and wastewater isolates, while antibiotic and metal resistance or virulence genes, were variable across the genomes and associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. The analysis of a larger and geographically more diverse group of genomes, suggested that antibiotic and metal resistance and virulence gene alleles were more prevalent and diverse in clinical isolates, while some quorum sensing, efflux systems and oxidative stress genetic determinants alleles were more prevalent and diverse in environmental isolates. The studies performed unveil a promising opportunity to implement comparable and reliable antibiotic resistance monitoring schemes. The harmonization of some procedures and the use of internal standards will enable worldwide comparisons of antibiotic resistance genes in wastewaters, and therefore improve and promote surveillance studies worldwide. The double evidence that antibiotic resistance features observed in clinical K. pneumoniae are maintained in the environment and that it is among the environmental isolates that stress dwelling features seem to be more diverse, supports the high capacity of K. pneumoniae for spreading through wastewater or in the environment, enhancing the risks of transmission back to humans.
| Date of Award | 28 Jan 2022 |
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| Original language | English |
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| Awarding Institution | - Universidade Católica Portuguesa
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| Supervisor | Célia Manaia (Supervisor), Isabel da Silva Henriques (Co-Supervisor) & Margarita Gomila (Co-Supervisor) |
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