Development of a Whole-Genome Sequencing (WGS) approach for the characterization of Legionella environmental isolates in water systems

Legionella is a Gram-negative bacterium that represent a public health issue, with heavy social and economic impact. Therefore, it is mandatory to provide a proper environmental surveillance and risk assessment plan to perform Legionella control in water distribution systems in hospital and community buildings. The thesis joins several methodologies in a unique workflow applied for the identification of non-pneumophila Legionella species (n-pL), starting from standard methods as culture and gene sequencing (mip and rpoB), and passing through innovative approaches as MALDI-TOF MS technique and whole genome sequencing (WGS). The results obtained, were compared to identify the Legionella isolates, and lead to four presumptive novel Legionella species identification. One of these four new isolates was characterized and recognized at taxonomy level with the name of Legionella bononiensis (the 64th Legionella species). The workflow applied in this thesis, help to increase the knowledge of Legionella environmental species, improving the description of the environment itself and the events that promote the growth of Legionella in their ecological niche. The correct identification and characterization of the isolates permit to prevent their spread in man-made environment and contain the occurrence of cases, clusters, or outbreaks. Therefore, the experimental work undertaken, could support the preventive measures during environmental and clinical surveillance, improving the study of species often underestimated or still unknown.

Chemiluminescence – based bioanalytical devices for astrobiological, food safety and clinical applications

At the intersection of biology, chemistry, and engineering, biosensors are a multidisciplinary innovation that provide a cost-effective alternative to traditional laboratory techniques. Due to their advantages, biosensors are used in medical diagnostics, environmental monitoring, food safety and many other fields. The first part of the thesis is concerned with learning the state of the art of paper-based immunosensors with bioluminescent (BL) and chemiluminescent (CL) detection. The use of biospecific assays combined with CL detection and paper-based technology offers an optimal approach to creating analytical tools for on-site applications and we have focused on the specific areas that need to be considered more in order to ensure a future practical implementation of these methods in routine analyses. The subsequent part of the thesis addresses the development of an autonomous lab-on-chip platform for performing chemiluminescent-based bioassays in space environment, exploiting a CubeSat platform for astrobiological investigations. An origami-inspired microfluidic paper-based analytical device has been developed with the purpose of assesses its performance in space and to evaluate its functionality and the resilience of the (bio)molecules when exposed to a radiation-rich environment. Subsequently, we designed a paper-based assay to detect traces of ovalbumin in food samples, creating a user-friendly immunosensing platform. To this purpose, we developed an origami device that exploits a competitive immunoassay coupled with chemiluminescence detection and magnetic microbeads used to immobilize ovalbumin on paper. Finally, with the aim of exploring the use of biomimetic materials, an hydrogel-based chemiluminescence biosensor for the detection of H2O2 and glucose was developed. A guanosine hydrogel was prepared and loaded with luminol and hemin, miming a DNAzyme activity. Subsequently, the hydrogel was modified by incorporating glucose oxidase enzyme to enable glucose biosensing. The emitted photons were detected using a portable device equipped with a smartphone's CMOS (complementary metal oxide semiconductor) camera for CL emission detection.