Colonization and disease induced by Streptococcus pneumoniae

The last couple of years our laboratory has been interested in how S. pneumoniae colonizes the nasopharynx and what mechanisms are required for these bacteira to transition from colonization to disease. S. pneumoniae is a formidable colonizer with a colonization rate betwen 20-90% in children depending on where in the world you measure and 5-50% in adults. This means that billions of people are currently colonized although few get sick. Epidemiologically, disease is associated with changes in the nasopharyngeal environment, with virus infection (influenza A virus, RSV, adenovirus etc) being the leading disease trigger. Disease can also be triggered by changes in teh microflora and other events leading to inflammation.


We have shown that S. pneumoniae colonizes the nasopharynx in the form of biofilms(Marks Infect Immun 2012) and have been ale to recapitulate biofilm formation in vitro using features of the nasopharyngeal environment such as a lower temperature (34 degrees C), an epithelial cell surface for attachment and a relative nutrient poor medium. Using this model system, S. pneumoniae make thick biofilms with intricate architectural features that are highly resistant to antibiotics after 48 hours. This is not found using higer ttemperature and abiotic surfaces for biofilm growth. Using this model the ability of clinical strains and mutant strains lacking factors involved in colonization to form biofilms correlate well with their ability to colonize animals, which is not shown in other biofilm models.

Figure 1. Biofilm formation on a bronchial carcinoma (A), healthy ciliated epithelial cells (B), in the nasopharynx of mice (C) and on abiotic surfaces (D).


We next showed that genetic exchange of fitness and antibiotic resistance occurs primarily in the colonizing biofilm and is about 1,000,000 times less effective during disease (Marks MBio 2012). This is true both during colonization in vivo and in our biofilm model in vitro. Using two different antibiotic-resistant strains we observed that approximately 1 in 1,000 bacteria would become double-resistant in biofilm/colonization models whereas only 1 in 1,000,000,000 bacteria would become double resistant during infection (sepsis; the classical model used by Griffith 1928 and Avery 1944). The implication for this has recently been shown also for other Streptococci that are known to carry the complete set of genes necessary for transformation but have not yet been seen to be competent. In collaboration with Micheal Federle and with the help of Don Morrison we have just succeeded in transformating Group A streptococci in biofilms (Marks J Inf Dis 2014).


Using influenza A virus as a disease triggerand our in vitro and in vivo models of biofilm colonization we were able to show that influenza A virus can cause an active release of bacteri from biofilms and that these actively dispersed organisms arehighly virulent in mice (Marks MBio 2013). The same active release occurs when biofilms are treated with environmental factors assocaited with virus infection. These include increased temperature (fever), increased concentration of extracellular ATP or nutrients (cell damage in the mucosa), or in the presence of nor-epinehrine (increased stress-response). This signalling where the bacteira are sensing the host environment is an exciting new field of study and these results show for the first time that specific molecules associated with virus infection cause release of virulent organisms that go on to cause infection of the middle ear (otitis media) and lungs (pneumonia) and can disseminate further into the bloodstream and cause sepsis.

Figure 2. Mice were colonized 48 hours with S. pneumoniae EF3030 and given Influenza A virus (IAV). The bacteiral burden in the tissues were measured 1 and 5 days after virus infection in nasopharyngeal tissue (tissue), nasopharyngeal lavage (lavage), lungs and in the middle ears (ears). IAV infection caused bacteira to move and cause infection in the lungs and middle ears but also increased the colonization in the nasopharynx.

Figure 3. Histopathology of the tissues after exposure to biofilm bacteria, bacteira actively dispersed from biofilms and broth-grown bacteria. As seen in the picture the infiltration of leukocytes (LI) was highly increased in the middle er (upper row) when mice were exposed to the Dispersed, biofilm released bacteria. Only a little inflammation was seen from broth-grown bacteira and no inflammation when biofilm bacteira were used. In the lungs (bottom row) no inflammation was seen in buffer treated mice or mice given biofilms whereas some inflammation was observed after broth-grown bacteira were inoculated (Planctonic). However, the inflammation and symptomatic disease was significantly increased in the presence of Dispersed bacteria. The phenotype of these bacteria are currently being investigated by a general transcriptome analysis using RNA-seq in collaboration with Melinda Pettigrew at Yale University School of Public Health.


Finally, we recently published a small study showing that biofilm bacteria can survive in dessicated form on surfaces for up to a month and that these bacteria are still infectious after dessication (Marks Infect. Immun. 2014). This is in contrast to broth grown bacteria that dies within a day or two. Biofilm bacteria could also survive much better on hands and when we visited a day care we were able to culture Streptococci from soft toys, books, crib linens and other hard surfaces, even after the children had not been there for a many hours. This indicates that S. pneumoniae and S. pyogenes may both be able to spread through surfaces, which is contrary to the current information form the CDC that says that spread only occurs through aerosolized droplets.