Genetic diversity and epidemiology of antimicrobial resistant Streptococcus pneumoniae isolates

Dissertation presented to obtain a Ph.D. degree in Biology, speciality in Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Infection with Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The main purpose of the studies in this thesis was to clarify the spread of antimicrobial resistant S. pneumoniae isolates recovered in Portugal. First we analysed antimicrobial resistance rates of invasive S. pneumoniae isolated from the Portuguese population. An increasing trend of S. pneumoniae isolates nonsusceptible to penicillin was observed until 2000. In the following years, the decline in -lactam nonsusceptibility was thought to be due to the introduction of the heptavalent pneumococcal conjugate vaccine (PCV-7) in Portugal in 2001. To shed some light on the impact of the introduction of the PCV-7 on the susceptibility of invasive S. pneumoniae to antimicrobial agents, we evaluated the association between IPD, serotypes and antimicrobial susceptibility in the paediatric population between 1999 and 2004. Our results suggest an increase in multidrug resistance after 2002, caused by a replacement of vaccine serotypes by nonvaccine serotypes among nonsusceptible isolates after the introduction of PCV-7. To provide useful information about the main factors involved in the selection of penicillin- and erythromycin-nonsusceptible invasive isolates, we evaluated the role of antimicrobial and vaccine use. The results obtained suggest that the use of macrolides was the main factor associated with an increase of penicillin- and erythromycin-nonsusceptible isolates. Furthermore, the heptavalent vaccine is failing to reduce antimicrobial resistance, as was expected, possibly because of the increased consumption of azithromycin. To a better understand of the emergence of erythromycin-nonsusceptible invasive S. pneumoniae isolates, we studied their genetic structure. We noted that the prevalence of macrolide-nonsusceptible S. pneumoniae isolates has increased in Portugal, from two routes: i) expansion of pre-existing nonsusceptible clones, and ii) importation of nonsusceptible clones mainly present in other European countries and their local diversification. Our results suggested that, in the Portuguese penicillin-nonsusceptible S. pneumoniae isolates that are closely related to the major Spanish clones, Spain9V-3 and Spain23F-1, the penicillin nonsusceptibility had spread by horizontal transfer of the pbp1A,penA and pbpX genes. We found higher divergence among the amino acid sequences of PBP2B, PBP2X and PBP1A from a collection of penicillin-intermediate invasive isolates than among those from highly resistant isolates. Finally, we demonstrate that serine/threonine kinase StkP of S. pneumoniae is involved in the bacterial response to penicillin and suggests that the activity of StkP allows the bacteria to bypass cell wall injury due to penicillin, while being independent from the pbp genes.