Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Novel approaches to microbial typing and their integration for the epidemiological investigation of healthcare-associated infection pathogens

Healthcare-associated infections (HAIs) represent a major public health concern, associated with significant morbidity, mortality, and economic burden worldwide. Their impact is further amplified by the increasing prevalence of antimicrobial resistance (AMR), particularly among Klebsiella pneumoniae and Acinetobacter baumannii, two of the most relevant opportunistic pathogens in intensive care units (ICUs). These microorganisms possess an extraordinary ability to persist in the hospital environment, colonize patients, and rapidly acquire resistance determinants through horizontal gene transfer. Their spread is sustained by a combination of clonal expansion, plasmid dissemination, and environmental reservoirs, making the reconstruction of transmission routes particularly challenging. A precise understanding of local and regional epidemiological dynamics is essential to detect outbreaks early, identify transmission pathways, and implement targeted infection prevention and control (IPC) strategies aimed at reducing the incidence of HAIs. This PhD project focused on developing and rationally integrating molecular and genomic typing methods to strengthen the epidemiological reconstruction of healthcare-associated pathogen transmission. Rather than relying on a single technique, the work aimed to define tiered strategies that balance resolution, cost, and scalability. Traditional typing methods such as PFGE and MLST, despite their limitations, remain valuable for rapid screening and lineage definition, especially in high-prevalence contexts. When combined with targeted or whole-genome sequencing, these approaches allow the progressive refinement of transmission networks, enabling early outbreak detection and a deeper understanding of pathogen evolution and spread. Several applied research studies illustrated how these methods can be effectively integrated in real-world epidemiological contexts. The first study demonstrated the value of combining PFGE and WGS during the SARS-CoV-2 pandemic to investigate A. baumannii outbreaks across multiple wards. PFGE enabled cost-effective large-scale screening of isolates, while WGS on a selected subset provided high-resolution insights into transmission dynamics and evolutionary relationships, optimizing both resources and epidemiological accuracy. A second study focused on the evolution of K. pneumoniae ST512 within a single patient, demonstrating how in vivo evolutionary events, including changes in the muco-viscosity phenotype, can occur during infection. This case highlights the challenges associated with national surveillance of hypervirulent K. pneumoniae, given the lack of standardized screening methods for this phenotype. It also underscores the critical role of genomic surveillance not only in outbreak detection but also in reporting complex and difficult-to-identify pathogen phenotypes. An additional line of work focused on emerging pathogens, notably Providencia stuartii. Here, the absence of established typing methods made WGS essential not only for outbreak reconstruction but also for creating and publishing the first MLST scheme for this species. The MLST scheme was deposited in PubMLST database, providing a new tool for future molecular epidemiology and surveillance efforts. To support operational prioritization in high-prevalence or resource-limited settings, a multiplex PCR assay was developed for rapid identification of prevalent K. pneumoniae sequence types circulating in Italy, enabling efficient triage of isolates for deeper genomic analysis Finally, the most innovative contribution of this work was the development of NanoTyping, a novel and cost-effective typing strategy for K. pneumoniae. This approach combines targeted amplification of key loci (MLST genes, wzi, and major carbapenemase genes) with Oxford Nanopore Technologies sequencing, enabling rapid and inexpensive inference of clonal lineage and resistance determinants. NanoTyping was validated during a real hospital outbreak, demonstrating its effectiveness in supporting real-time molecular epidemiology and outbreak management, particularly in high-prevalence and low-resource settings. Overall, this work demonstrates that strengthening microbial epidemiology depends on designing rational, complementary workflows rather than selecting a single “best” technique. Integrating methods with different depth and scalability allows precise, timely reconstruction of transmission chains and supports more effective IPC measures. By developing tools such as the multiplex PCR, the first P. stuartii MLST, and NanoTyping, this thesis contributes practical and scalable solutions to improve surveillance and control of multidrug-resistant organisms in healthcare environments. Improving the precision, speed, and cost-effectiveness of outbreak investigation workflows, is an essential goal to reducing the incidence and impact of HAIs. Strengthening pathogen typing and genomic epidemiology represents a cornerstone for infection prevention strategies, enabling health systems to detect transmission events earlier, respond more effectively, and ultimately curb the spread of multidrug-resistant organisms in healthcare settings.