New insights into the pathogenicity of Listeria monocytogenes clonal complexes

Abstract

Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen mainly affects specific vulnerable groups such as the elderly, newborns, pregnant women, and immunocompromised individuals. The severity of the illness varies based on both intrinsic factors of the affected individuals and the pathogen itself. Therefore, different clinical outcomes can be expected, ranging from simple gastroenteritis to central nervous system, maternal-neonatal, and bacteremia infections. Furthermore, L. monocytogenes is genetically and phenotypically diverse species, resulting in a high variation in stress response and virulence potential among strains. Multilocus sequence typing (MLST) has been widely adopted as a reference method to categorize the clonal structure of bacterial species and to define clonal complexes (CCs) of genetically related isolates, i.e., derived from the same ancestor. In L. monocytogenes, the combination of MLST and epidemiological data allows to distinguish hypervirulent CCs, which are notably more prevalent in clinical cases and typically associated with severe forms of the disease. Contrarily, other CCs, denominated hypovirulent are predominantly isolated from food and food processing environments, and they are linked with the occurrence of listeriosis in highly immunosuppressed individuals. The aim of the present work was to assess and compare the virulence potential of L. monocytogenes strains belonging to both hypervirulent (CC1, CC2, CC4, CC6, CC87 and CC388) and hypovirulent CCs (CC9 and CC121). Sixteen isolates collected from clinical cases occurred in Portugal, distributed among eight distinct CCs, were selected and characterized in terms of: i) in vitro invasion efficiency in Caco-2 cells; ii) presence of premature stop codon (PMSC) mutations in the inlA gene; iii) in vivo infection in Galleria mellonella larvae and health index score characterization, iv) hemolytic activity; and v) ability to survive to the acid barrier of the stomach (pH 2.5 and 3). Five strains were further characterized in terms of immunomodulatory effect in infected G. mellonella larvae and expression of five virulence genes (inlA, inlB, actA, hly, plcA, and prfA). The results obtained demonstrated that there is a clear-cut distinction in the invasion efficiencies in Caco-2 between strains belonging to hyper- and hypovirulent CCs, with CC121 and CC9 isolates showing impaired invasion ability and PMSCs in inlA. Although this CC-related evidence was found in in vitro assays, isolates from CC9 showed an augmented virulent phenotype upon infection of G. mellonella larvae, while some isolates from hypervirulent CCs (CC2 and CC6) yielded reduced larvae mortality rates. Additionally, intra-clonal complex differences were observed among CC6 strains. Comparison of haemolytic activity among CCs, performed using classical blood agar plates and a microplate assay, showed that a CC121 strain presented higher ability to lyse red blood cells. This observation was validated by higher expression levels of hly (the gene encoding the protein listeriolysin O, LLO); this strain also exhibited increased expression of the virulence genes inlA and actA. No significant differences were found between the CCs concerning survival at low pH. In conclusion, the present study emphasizes the importance of incorporating both genotypic and phenotypic data when characterizing the virulence potential of L. monocytogenes. However, further research, such as genome analysis, may shed some light and explain some of the reported phenotypes. Moreover, as the zero-risk infection with L. monocytogenes is still not yet achievable, reliable biomarkers for risk assessment in the food industry need to be identified.

Date of Award	23 Jan 2024
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Vânia Borges Ferreira (Supervisor), Paula Teixeira (Co-Supervisor) & Rui Magalhães (Co-Supervisor)