Increasing evidence suggests that microbial biofilms play a role in chronic human infections.7,9,10,19,20
Criteria proposed for characterizing biofilm infections include direct examination of an infected tissue revealing pathogenic bacteria in clusters within a matrix attached to a surface, infections localized to a particular anatomical site, and evidence of recalcitrance to antibiotic treatment despite antibiotic sensitivity demonstrated by planktonic forms.20
In this investigation, we observed biofilms characterized by bacteria in matrix-enclosed adherent clusters in 46 of 50 evaluable specimens (92%) from children with chronic localized middle-ear disease undergoing TT placement who had not responded to multiple courses of antibiotics, thus fulfilling all 3 biofilm diagnostic criteria.
CLSM imaging revealed clusters of bacteria on the MEM of patients with both OME and recurrent OM. Since specimens from those with recurrent OM lacked accompanying effusions for PCR and culture, in situ pathogen-specific identification methods were developed to further characterize these biofilms. CSLM imaging, using both antibody and FISH probes specific for pneumococcus, also identified coccal bacteria within matrices on the pediatric MEM specimens that corresponded morphologically with pneumococcal biofilms imaged in vitro18
and on chinchilla bullar epithelium (data not shown), supporting the hypothesis that pneumococcus forms biofilms during chronic infections.
All of the specimens that demonstrated pneumococcus were rated as positive by at least 2 independent diagnostic modalities, suggesting that the prevalence of S pneumoniae
is high in chronic OM. This percentage is higher than the number of PCR-positive pneumococcal specimens found in this and other studies4
; however, because PCR is performed only when an effusion is present, this method does not assess recurrent OM. Middle-ear mucosa specimens in the current study also revealed a high prevalence of biofilms that were FISH 16S–positive for H influenzae.
The H influenzae
observed in these biofilms assumed a coccobacillary form, similar to those forms observed in experimental H influenzae
and distinct from the pure bacillary form seen during planktonic growth.
Fluorescence in situ hybridization has been demonstrated to be a specific and sensitive tool for the assessment of bacteria in clinical samples and has proven useful for providing spatial and morphological data unobtainable by PCR14
; however, there are technical issues associated with FISH-based imaging. First, specimen preparation requires stringent wash steps, which undoubtedly remove a significant fraction of any biofilm. Second, when using small biopsy specimens that cannot be subdivided, there is a maximum of 2 species that can be evaluated per specimen when also using the eubacterial probe, because only 3 fluorescent dyes are available for FISH-based analyses. Notwithstanding these difficulties, in all but 1 case for which positive FISH results were obtained, species-specific corroborating data were obtained using other methods.
In this study, the in situ assessment of MEM biopsy specimens using both generic and species-specific bacterial probes is supportive of a biofilm etiology for chronic OM. The vast majority of MEM specimens obtained from children undergoing TT placement evidenced matrix-enclosed adherent bacteria, whereas control specimens from patients undergoing cochlear implantation had no evidence of bacteria. Moreover, these data suggest that the bacterial species involved in these mucosal biofilms are the same pathogens commonly associated with OM. However, because of the small amount of tissue and the inability to simultaneously perform positive and negative 16S controls on each specimen, we cannot entirely exclude the possibility of nonspecific interactions with the probes used in these analyses. We attempted to address this limitation by using a larger number of tests in the aggregate than could be performed on each specimen. Moreover, in vitro evaluations of all of the 16S and antibody probes used in this investigation identified no cross-reactivity when tested against a battery of clinical strains representing the common human mucosal pathogens. A second limitation of this investigation was the small number of age-matched controls who were available for participation in the study; to partially compensate for this difficulty, we expanded the age range for obtaining control specimens.
Collectively, the data presented in this study support the hypothesis that biofilms may play a role in the pathogenesis and chronicity of OME and also provide evidence that at least some mucosal specimens from patients with recurrent OM harbor bacterial biofilms. This finding is surprising in light of the body of literature that suggests that consecutive episodes of acute OM are associated with unique bacterial strains.21
Our findings are more consistent with recurrent OM as a chronic disease with episodic acute exacerbations. Leibovitz et al22
have demonstrated that approximately 30% of cases of recurrent OM result from relapses attributable to the original organism. The true relationship between bacterial persistence demonstrated on biopsy and the clinical observation of a disease-free period clearly requires further investigation.
Importantly, the findings from our study do not exclude other potential pathogenic factors associated with OME, such as an antecedent viral upper respiratory infection, eustachian tube dysfunction with impaired gas exchange, a genetically predisposed host, persistent inflammatory mediators, or exacerbation by gastroesophageal reflux. However, these findings do argue against the notion that OME is the result of a non-bacterial inflammatory process and also indicate that equating culture negativity and absence of bacteria is incorrect.
The finding that biofilms are present in almost all cases of OME in this study may help to explain the lack of antibiotic efficacy for this disorder, given that biofilm bacteria are more antibiotic resistant than single cells in suspension.23–28
This resistance may stem from the fact that oxygen and nutrient limitation within biofilms induces metabolic quiescence, which in turn reduces antibiotic effectiveness.2,23
Other evidence suggests that biofilm bacteria may have genetic mechanisms, selected for in the biofilm, that provide antimicrobial protection.29–31
In addition, the biofilm provides a physical barrier that enhances pathogen resistance to host defenses such as opsonization, lysis by complement, and phagocytosis.7