Streptococcus pneumoniae, commonly called pneumococcal , is a pathogenic bacteria found in the human nasopharynx (especially in girls and boys) and can cause mild diseases, such as otitis or sinusitis, to more severe pathologies, such as pneumonia and meningitis. Although there are vaccines to prevent infections caused by this pathogen, as well as different antibiotics to treat them, it is estimated that these types of diseases cause more than one million deaths annually worldwide. This situation makes pneumococcus the pathogen that causes the most deaths among those that cause infectious diseases preventable by vaccines. The effectiveness of the different antibiotics available to treat pneumococcus is currently critically compromised by the appearance of different strains that develop tolerance, persistence or resistance to certain pharmacological treatments. In this sense, the existence of tolerance mechanisms to vancomycin, as well as resistance to penicillin and the group of antibiotics known as fluoroquinolones, has already been established.
Recently, scientists from CONICET presented research in which they show, based on tests in cellular models, that exposure to intracellular oxidative stress could lead small populations of pneumococcus to develop mechanisms of persistence to treatment with fluoroquinolones. Persistence is a type of tolerance to high concentrations of antibiotics that a bacterial subpopulation acquires from environmental factors and that is not inherited to offspring (that is, it does not involve modifications in the genome). On the other hand, the persistence of pneumococcus to fluoroquinolones is clinically relevant, above all, because antibiotic therapy could fail due to the emergence of strains with resistance to this type of antibiotics. In this regard, it is considered that persistence may be a step prior to the appearance of cases of antibiotic resistance .
The study, led by José Echenique , CONICET researcher at the Center for Research in Clinical Biochemistry and Immunology (CIBICI, CONICET-UNC), was published in the journal Microbiology Spectrum of the American Society of Microbiology. The first author of the study was Mirelys Hernández-Morfa , a Council doctoral fellow at CIBICI.
The intracellular life of pneumococcus
Although pneumococcus lives mainly in the extracellular environment, it can remain inside cells of the immune system or pneumocytes (specialized cells that form the lung alveoli) temporarily for several hours. “When a bacteria enters a cell of the immune system, such as macrophages or neutrophils, a burst of oxidative stress is unleashed that aims to kill the pathogen. That is, the cells begin to produce hydrogen peroxide, among other reactive oxygen species, to get rid of the infection,” explains Echenique.
Now, although the majority of pneumococcus bacteria die due to the hydrogen peroxide attack, it has been established that there is a subpopulation that manages to survive. “Pneumococcus is a bacteria that has strong resistance mechanisms against oxidative stress, given that it needs to survive the hydrogen peroxide itself that it usually produces to compete with other bacteria that inhabit the nasopharynx, which is lined by about seven hundred different microorganisms. If pneumococci did not have enzymes that can degrade hydrogen peroxide at the intracellular level, there would not be a subpopulation with the possibility of surviving the oxidative burst that occurs when they enter a human cell. Being able to survive these levels of oxidative stress is already a fairly sophisticated mechanism for an organism that is normally extracellular,” says the researcher.
Development of persistence to fluoroquinolones
Fluoroquinolones are a group of antibiotics used to treat infections caused by pneumococcus, whose mechanism of action is also linked to increased levels of oxidative stress. Knowing that prior exposure to hydrogen peroxide induces the defense mechanism of the pneumococcus against this type of compounds, the authors proposed that this scenario could counteract the effect of fluoroquinolones. This led the research team led by Echenique to analyze whether exposure to hydrogen peroxide led the pneumococcus to develop persistence mechanisms to fluoroquinolones, and they corroborated this through tests in culture media and with different cellular models.
“To determine if exposure to oxidative stress led it to develop persistence to antibiotics, the first thing we did was expose the pneumococcus to a culture medium with similar amounts of hydrogen peroxide to those that could be exposed inside an immune system cell. . By adding fluoroquinolones we saw that the survival rate of these bacteria against this antibiotic was actually increased, compared to what happened with pneumococci that had not been exposed to oxidative stress. We also managed to identify some genes that are involved in the development of this mechanism,” says Echenique.
The next step was to test whether this behavior could be reproduced in cellular models, such as macrophages, pneumocytes and neutrophils.
“In cellular models, as we already knew, the entry of the pneumococcus generates an increase in the levels of hydrogen peroxide inside the cells, which leads to a high percentage of the bacteria dying, although a subpopulation is able to survive. After several hours, we added fluoroquinolones to see if exposure to intracellular oxidative stress also produced the development of a persistence mechanism for this antibiotic, as had occurred in the assays in culture media. Thus, we were able to identify that the induction of the fluoroquinolone persistence mechanism also occurs when the pneumococcus is exposed to intracellular oxidative stress when it invades cells,” the researcher develops.
Echenique highlights that this type of tolerance does not mean resistance to antibiotics, because for this there must be mutations in certain genes or the incorporation of new genes. “When you take away the hydrogen peroxide and treat them again with fluoroquinolones, the persistent bacteria regain sensitivity to the antibiotics and die.”
Furthermore, Echenique clarifies: “This mechanism of persistence to antibiotics is relevant from a clinical point of view, since it would reduce the efficiency of antimicrobial treatment in patients with pneumococcal infections, and, on the other hand, greater exposure to fluoroquinolones would facilitate the generation of resistant strains.”
