In this study, we sought to test the hypothesis that GAS colonizes the middle ear in a biofilm. The chinchilla animal model has been a useful tool for studying in vivo
biofilm formation (2
). Recently, the chinchilla model was used to investigate Streptococcus pneumoniae
biofilm formation in vivo
). In that study, middle ear effusion and inflammation characteristic of OM were observed along with macroscopic structures that were indistinguishable by the naked eye from the structures observed in this study (38
). Similar effusion, inflammation, and structure characteristics were also observed following infection with Haemophilus influenzae
). Thus, the formation of these structures appears to often accompany the onset of otitis media in this model; however, our data indicate that the presence of these macroscopic structures does not determine the distribution of the infecting organism within the structure. That is, the pathogen may be aggregated into microcolonies or biofilms or may be more randomly distributed throughout the structure and the accompanying effusion.
In the S. pneumoniae
study, biofilm formation correlated with resistance to clearance by the host immune system (38
). Given that MGAS5005 Δsrv
does not form biofilms in vitro
), we hypothesized that we would see decreased biofilm formation by MGAS5005 Δsrv
and an increased rate of clearance in vivo
. While we did note that biofilm formation was decreased or absent, animals infected with MGAS5005 Δsrv
had higher bacterial loads in middle ear effusions until 7 dpi and significantly higher bacterial loads in the effusion at 2 and 4 dpi (Fig. ). Furthermore, animals infected with MGAS5005 Δsrv
had a lower rate of clearance of the organism from the effusion than those infected with MGAS5005 (Fig. ). While the data did not reach statistical significance, the rate of mortality for those animals infected with MGAS5005 Δsrv
was also greater (Fig. ).
In contrast, a different phenotype was observed in the MGAS5005-infected samples. Plate counts of the homogenized macroscopic structures revealed the presence of MGAS5005 in all 8 of the experimentally infected ears at 7 dpi (Fig. ). A combination of SEM with LIVE/DEAD and Gram staining revealed the presence of three-dimensional aggregates of cells or microcolonies indicative of biofilms within the macroscopic structures removed from the middle ears of MGAS5005-infected samples. For GAS, evidence of biofilm formation as dense clusters of bacteria or microcolonies in zebrafish muscle tissue has been previously presented (6
). Microcolonies have also been previously presented as evidence of GAS biofilms on human cells in vitro
). These microcolonies were largely absent in the analyzed MGAS5005 Δsrv
-infected samples. Rather, the MGAS5005 Δsrv
organisms were randomly distributed throughout the macroscopic structure and dispersed into the effusion. This is perhaps best shown in the LIVE/DEAD stained samples (Fig. ), in which living MGAS5005 cells were clustered into a densely packed three-dimensional core compared to the randomly distributed viable MGAS5005 Δsrv
organisms. This is not to say that some clustering of MGAS5005 Δsrv
was not observed, but it was found in dispersed form far more frequently than MGAS5005. Thus, the MGAS5005 Δsrv
structures are less adherent, less organized, and less stable. These results do not contrast with our in vitro
); rather, the in vivo
data suggest that host components may partially rescue a biofilm-deficient mutant and likely add to the overall biofilm structural stability of a biofilm-proficient strain (21
). We conclude that the increased number of CFU of MGAS5005 Δsrv
found in the effusion were due to the decreased biofilm phenotype exhibited by this strain. Furthermore, the increase in CFU of MGAS5005 found in the effusion at 7 dpi may reflect a population density cue that signals dispersal into the effusion. We are continuing to investigate this hypothesis.
In our in vitro
work examining GAS biofilm formation, we presented evidence that the significant deficiency in the ability of MGAS5005 Δsrv
to form biofilms was due to constitutive production of SpeB. SpeB has previously been shown to degrade GAS proteins such as M protein and DNase (5
) as well as host proteins, including complement, antibodies, and extracellular matrix components (7
). Further work has supported a role for SpeB in dissemination, tissue damage, and prevention of phagocytosis by polymorphonuclear leukocytes (25
). In addition, patients suffering from severe, invasive GAS disease have decreased levels of antibodies to SpeB compared to healthy counterparts, suggesting that individuals with low anti-SpeB antibody titers may be at a higher risk for developing severe disease (20
). Thus, if our hypothesis is correct, allelic replacement of speB
in the MGAS5005 Δsrv
background should restore biofilm formation. SEM and Gram staining revealed the presence of biofilms within the macroscopic structures recovered from MGAS5005 Δsrv
-infected animals (Fig. ).
Taken together, we believe this work is significant on three counts. First, we have demonstrated a model for the study of GAS otitis media. While the chinchilla model itself is not new, we are unaware of GAS being used in this model previously. Transbullar infection with GAS resulted in inflammation, including vessel dilation, fluid accumulation, and opacity of the tympanic membrane associated with otitis media and similar to what has been previously described for other organisms (2
). It is important to continue to develop this model to determine whether intranasal infection can lead to GAS OM. Alternatively, we are currently developing this model to study the advancement of GAS OM to the more severe disease stage of mastoiditis in an effort to understand how GAS biofilms or biofilm dispersal may be involved in this process.
Second, we have provided evidence that GAS naturally forms biofilms during otitis media infection and that these biofilms may contribute to persistence within the macroscopic structures that form as a result of the disease and immune response. However, through the MGAS5005 Δsrv data, we have also demonstrated that biofilms are not required for infection or overt disease following transbullar inoculation. Thus, it appears that GAS can exist at the site of infection in either a biofilm or nonbiofilm state. Longer time courses of infection are needed to determine whether or not the GAS biofilm does provide a resistance to overall clearance from the middle ear, but it appears that, based upon increased bacterial loads in the effusion and increased mortality, the nonbiofilm state, as seen in the MGAS5005 Δsrv-infected animals, is more virulent.
Finally, the ability of GAS to exist in a biofilm or nonbiofilm state during an active infection strongly suggests that there is a mechanism for the dispersal of the biofilm. Our previous in vitro
work and the genetic evidence presented here support one hypothetical mechanism by which Srv-mediated regulation of SpeB contributes to biofilm stability or dispersal and dissemination (14
; Roberts, submitted). During colonization and biofilm maturation, Srv-mediated control, whether direct or indirect, of speB
/SpeB is tightly regulated, allowing little to no production of SpeB. Upon the sensing of some environmental signal(s), control is relaxed, SpeB is produced, and the biofilm is dispersed, allowing dissemination and possible disease transmission. We are continuing to explore this hypothesis.