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Unravelling the mechanisms of competence induction by antibiotic stress in Streptococcus pneumoniae

Periodic Reporting for period 1 - PneumoCompetence (Unravelling the mechanisms of competence induction by antibiotic stress in Streptococcus pneumoniae)

Reporting period: 2015-05-01 to 2017-04-30

Despite the introduction of several vaccines, the human pathogen Streptococcus pneumoniae remains one of the leading bacterial causes of mortality worldwide, killing 1 million of children each year. In developed countries, the rise of multi-drug resistance in S. pneumoniae during the last decade is a serious cause of concern, since the patient is more likely to require hospitalization consuming high health resources, and increasing the risk of death.
The main mechanism by which S. pneumoniae acquire and spread antibiotic resistance is by activation of the competence state that allows the uptake of exogenous DNA. Strikingly, competence is induced by several antibiotics, but the molecular mechanisms driving competence development are poorly understood.
Using several approaches, I identified several common antibiotics that induce competence and thereby promotes the acquisition and spread of antibiotic resistance. One particular interesting case is the effect of the betalactamase inhibitors (clavulanic acid and tazobacta) not active against Streptococcus pneumoniae (pneumococcus does not produce betalactamases), but still able to promote competence. Clavulanic acid has no antimicrobial activity using clinical concentrations, and is administered with amoxicillin (which alone is not able to induce competence) for the treatment of infections caused by bacterial producers of betalactamases, mainly Haemophillus influenzae, Moraxella catarrhalis or Pseudomonas aeruginosa. This is a severe cause of concern because the combination of amoxicillin-clavulanic acid is the second most antibiotic sold during the last years (Weber F et al, ISM Health Database 2016), and is included in clinical guidelines and recommendations for the treatment of respiratory infections (i.e. bacterial sinusitis, acute otitis media, community-acquired pneumonia and acute exacerbations of chronic bronchitis), in most of which S. pneumoniae is the main etiological agent (19). Hence, the combination of amoxicillin-clavulanic acid for the treatment of pneumococcal infections has no extra benefit but promotes the acquisition of antibiotic resistance; thereby, the guidelines should be reviewed and, when the aetiological agent of the infection is S. pneumoniae, the patient should be treated with amoxicillin alone instead to the combination with clavulanic acid.
In parallel, I have identified small molecules inhibitors of the competence and transformation processes, which could help for future novel treatment strategies against bacterial infections and the mitigation of the spread of antibiotic resistance.
During this first year of the project, I have tested the competence induction by antibiotic stress. A total of 86 common antibiotics from different classes were tested: betalactams, fluoroquinolones, aminoglycosides, MLSB group (macrolides, lincosamides and streptogramines), tetracyclines, etc. Of them, clavulanic acid and aztreonam were selected for further studies since they were the most promising hits (widely used on the clinical treatment of respiratory infections).
Microarrays experiments revealed no significant changes on gene expression in presence of either short (15min) or long (3h) exposure to the antibiotics. These results confirms that the mechanism of competence induction is post-transcripcional, in contrast to the fluoroquinolones mechanism, previous described (Slager et al, Cell2015).
Then, I also performed microscopy experiments, which revealed the formation of chains in presence of the compounds, without other phenotypic changes (no disruption of membrane, cell wall or cell division machinery). Extra experiments with different medium viscosity suggest that the presence of chains as a result of the antibiotic effect, would reduce the diffusion of the CSP1, enhancing the interaction between peptide and receptor (ComD). In order to confirm this hypothesis, I am working on the mechanism trough these antibiotics induce the chain formation. A publication in a format poster was presented in the New Approaches and Concepts in Microbiology, from 11-14 October 2015 in the EMBL, Heidelberg, Germany (poster attached as fig.1)
Once unraveled, I will prepare the first manuscript.

The second part of the project (WP4), aimed to seek inhibitors of competence that would reduce the acquisition and spread of antibiotic resistance. For this purpose, I performed the Secondment at the Typas lab in the EMBL of Heidelberg (for more info, see the proposal). A total of 1300 compounds were tested and we found 48 compounds that were able to inhibit competence without affecting the viability of the pneumococci (fig2). Those compounds belong to two different classes, and I have selected one of each class in order to confirm the HTS results. Both compounds are able to block spontaneous competence development, the horizontal gene transfer between two different pneumococcal strains, and also the uptaking of naked DNA added to the medium. I am performing several experiments in order to unravel the exact mechanism through these compounds act.
The fact that clavulanic acid, a compound not active against S. pneumoniae, is able to induce competence, together with the knowledge of the mechanism of action, may have an impact on the clinical treatment of respiratory infections. So far, amoxicillin-clavulanic acid is the most used empirical combination when the clinical unknown the bacteria causing the infection. Based on our results, there is no need to administrate amoxicillin-clavulanic acid in a patient with a pneumococcal infection, because the risk of acquire antibiotic resistant genes is much higher than the treatment with amoxicillin alone. Further in vivo experiments are needed to confirm our results, but these can have a great impact on the reduction of antibiotic resistance in this pathogen.
About the second aim, we are about to patent the compounds as inhibitors of competence. The idea is to use those compounds as co-adjuvants of the antibiotics (in the same pill) in order to reduce the acquisition of resistance by the commensal pneumococci which resides harmfully in the nasopharynx of the patient. Once the patent is published, a manuscript will be send to a high impact journal.
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