Next Generation Sequencing-based detection and characterization of microbial pathogens causing invasive infections and outbreaks in ICU: towards improved management of the high-risk patient

Healthcare-associated infections (HAIs), often exacerbated by resistance to last-resource antimicrobials (AMR), are a major global threat, as documented by the new European Recommendation on Combating AMR. Considering the state-of-art scientific innovations and common issues (time, cost) related current diagnostic, the goal of this project is to develop a molecular approach for the detection and characterization of microorganisms causing invasive infections and outbreaks in ICUs and surgery using WGS. During the project, different microorganisms (bacteria and mycetes) were selected for WGS performed adopting two different approaches: i) long-read sequencing, using the Oxford Nanopore MinION (ONT, Oxford, UK) ii) short-read sequencing, using Illumina platforms in outsourcing. In this project, WGS, was applied in four key fields: 1. Novel strategies for pathogen identification: in two invasive infections mediated by Gordoniae terrae and Salmonella tiphy, not identifiable by standard methods, WGS enabled rapid pathogen identification, demonstrating how the introduction of this technology in the clinical microbiology laboratory can play an important role in supporting diagnosis and appropriate treatment, improving patients’ outcome. 2. Typing of microbial agents of Public Health concern: M. bovis Bacillus Calmette-Guérin (BCG) is a strain of mycobacterium closely related to M. tuberculosis, which can be administered intravesically for the treatment of transitional cell carcinoma of the urinary bladder. Nevertheless, BCG immunotherapy has been associated with some adverse reactions, that can be observed after months or years. Long-read sequencing of strain BCG-GE, isolated from a patient admitted to a tertiary-care hospital in Albenga (Savona, Italy), previously subjected to BCG instillation with the OncoTice strain (Merck, US), yielded a complete genome and allowed identified as M. tuberculosis var. bovis BCG and a global phylogenetic analysis including other BCG strains. Results showed that BCG-GE was highly related with the BCG Tice strain, commonly employed in immunotherapy practices, demonstrating that the vertebral osteomyelitis was most an adverse event following BCG instillation. 3. Tracking of nosocomial outbreaks and genomic surveillance: the WGS-based tracking of a difficult-to-control nosocomial outbreak of Candida auris in San Martino Hospital (HSM), where it first emerged in 2019, revealed that all cases belonged to the clade I (South Asian), and that some putative cases no longer occurred as part of a defined outbreak. Typing of antifungal resistance markers revealed that resistance to echinocandins likely emerged following selection of FKS1S639F and FKS1F635Y mutants upon prolonged exposure to caspofungin and/or anidulafungin. 4. Detection of novel antimicrobial resistance mechanisms: WGS-based typing of a Klebsiella pneumoniae strains (NE 368) of clinical origin, testing resistant to ceftazidime/avibactam, revealed the presence of a blaKPC gene encoding a novel 270-loop mutated variant of KPC-3 (KPC-109). Cloning experiments demonstrated that KPC-109 production mediated resistance/decreased susceptibility to avibactam-based combinations (with ceftazidime, cefepime, and aztreonam) and cefiderocol, with a trade-off on resistance to carbapenems. However, in the presence of porin alterations commonly found in high-risk clonal lineages of K. pneumoniae, KPC-109 was able to confer clinical-level resistance to carbapenems. These findings expanded the current knowledge of the diversity of emerging variants of the KPC enzyme with 270-loop alterations that can be found in the clinical setting. In conclusion, WGS is a key tool in many areas of clinical microbiology, providing microbiologists and clinicians with rapid and valuable information not available by other commercial methods. To date, however, several limitations to the application of WGS in clinical microbiology persist, for which the near future envisions the integration of library preparation, sequencing, and data analysis into a single efficient workflow, allowing implementation in the laboratory routine.