Epidemiology, population structure, and pathogenesis of opportunistic respiratory tract colonising bacteria

Abstract

[eng] The bacterial colonisation of the respiratory tract is critical for health as it establishes a symbiotic relationship between the bacterial community and the host. Haemophilus influenzae and Staphylococcus aureus are two opportunistic microorganisms that usually colonise the respiratory tract, and while their interaction is usually asymptomatic, in some cases colonisation is the first step before the development of severe infections. From a microbiological perspective, the goals of this thesis focus on studying the epidemiology, the population structure, and the pathogenesis of these microorganisms, since changes in some of these factors may influence the development of serious diseases. We provided an update on H. influenzae disease at the Hospital Universitari de Bellvitge between 2014 and 2019, and compared its evolution to a previous period, 2008 and 2013. The overall incidence of H. influenzae disease was 2.07 cases per 100,000 population, and remained constant between the two periods. During the second period (2014-2019), the rate of ampicillin resistance increased from 10% to 17.6%, mainly due to the production of β-lactamase TEM-1. Non-typeable H. influenzae (NTHi) strains were the leading cause of invasive H. influenzae disease in both study periods (84.3% and 85.3%, respectively). NTHi population was genetically heterogeneous, and its classification into six clades (I-VI) based on the presence and absence of 17 accessory genes showed that most of them were clustered in clade V. Strains from clade V and grouped the most frequent NTHi STs (ST3, ST103, ST160), and were associated with the production of β-lactamase TEM-1. On the other hand, capsulated strains were uncommon in invasive disease, with serotype f being the most prevalent (8.8%). We analysed the population structure and the pangenome of capsulated strains, paying especial attention to serotype f, which is rising among cases of invasive disease in Europe. Capsulated genomes belonged to a small number of clonal complexes (CCs) associated with each serotype, which were highly clonal. The pangenome analysis revealed that serotype b genomes had a larger pool of genes and more accessory genes per genome than the other serotypes. The analysis of single nucleotide polymorphisms (SNPs) showed that the total number of SNPs in serotype f was significantly lower than serotypes a, b, and e, indicating low variability within CCs of serotype f. These findings support the high genetic stability of this serotype observed in the strains isolated from different countries, revealing no relationship between phylogeny and geographical origin. We also examined the genetic changes of H. influenzae during respiratory colonisation, which could contribute to the progression of chronic obstructive pulmonary disease (COPD). We studied the respiratory isolates from 15 patients with COPD and prolonged azithromycin therapy, of which, four had persistent H. influenzae colonisation, and two were persistently colonised by H. parainfluenzae. All persistent lineages isolated before treatment were azithromycin susceptible, but antibiotic pressure led to resistance development in the first months. Different resistant genetic determinants were identified in each lineage, including successive mutations in 23S rRNA, mutations in the genes encoding ribosomal proteins L4 and L22, and the acquisition of MefE and MsrD efflux pumps. Other genetic variation mainly occurred in genes associated with cell wall and inorganic ion transport and metabolism, such as licA and hgpB genes, linked to lipooligosaccharide synthesis and heme uptake, respectively. Regarding S. aureus, we compared the biofilm formation of endemic (CC5, CC8, and CC22) and sporadic (CC1, CC30, CC45, CC72, and CC398) methicillin-resistant S. aureus (MRSA) clones in our healthcare setting. The endemic clones produced more biofilms than the sporadic clones. Furthermore, allelic variant 1 of sasG, encoding the S. aureus surface protein G, was found in the most prevalent clones (CC5 and CC8), in correlation with increased biofilm formation. Thus, allelic variant 1 of sasG could be considered an important virulence factor that may favour bacterial survival. Furthermore, we also examined the genetic changes associated with intracellular invasion in patients with respiratory colonisation and subsequent bacteraemia caused by S. aureus. The bacteraemic isolates showed a significantly lower ability to invade lung epithelial cells than the respiratory isolates, and genetic analysis revealed differences in genes encoding proteins involved in the interaction of epithelial cells with the bacterial surface.

[cat] La colonització bacteriana del tracte respiratori és essencial per la salut per establir una relació simbiòtica entre la comunitat bacteriana i l’hostatger. Haemophilus influenzae i Staphylococcus aureus són dos dels microorganismes oportunistes que habitualment colonitzen el tracte respiratori, i mentre la seva interacció normalment és asimptomàtica, aquesta colonització pot ser el pas previ en el desenvolupament d’infeccions greus. Aquesta tesi es centra en l’epidemiologia (Estudi 1), l’estructura poblacional (Estudis 1 i 2) i la patogènesi (Estudis 3 i 4, i Resultats Addicionals) d’aquests microorganismes, assumint que canvis en alguns d’aquests factors poden influenciar el desenvolupament de malalties greus.