INTRODUCTION: Between 20% and 50% of middle ear effusions in otitis media with effusion test positive for bacteria, i.e. H. influenzae, M. catarrhalis and S. pneumoniae. In one study 48% of effusions that tested negative by culture wre positive by polymerase chain reaction (PCR).1 This has been advanced as evidence for the presence of bacterial biofilms, as agents leading to the persistence of glue ear. There is, however, the possibility that the DNA detected is the fossilized remains of bacteria from previously cleared infections. If this is the case, then the effusioin must in some way be protecting the DNA from breakdown by DNases. METHODS: Here we demonstrate, using a viscosity assay, that middle ear effusions taken from children during myringotomy inhibit the breakdown of DNA in a concentration-dependent manner. Middle ear effusion homogenates 0.5-3.0 ml (1 : 10 Vol. vol/effusion: PBS) were incubated with 3 ml of DNA (0.5 mg/ml in PBS) plus 0.2 ml of DNase 1 (500 Kunitz) at 37 degrees C for 24 h. Viscosity measurements were taken at regular intervals and the changes in viscosity expressed as a percentage of time 0. DNase activity was inhibited by 1.5 ml and 3.0 ml of effusion 48% and 91%, respectively, after 30 min incubation. CONCLUSION: This study demonstrated that effusions have the ability to inhibit nuclease activity, and the reported presence of non-culturable bacteria based on DNA detection by PCR could still represent fossilized remains and not viable bacteria. The same could also be true of recent reports of bacterial mRNA.2
INTRODUCTION: Between 20% and 50% of middle ear effusions in otitis media with effusion test positive for bacteria, i.e. H. influenzae, M. catarrhalis and S. pneumoniae. In one study 48% of effusions that tested negative by culture wre positive by polymerase chain reaction (PCR).1 This has been advanced as evidence for the presence of bacterial biofilms, as agents leading to the persistence of glue ear. There is, however, the possibility that the DNA detected is the fossilized remains of bacteria from previously cleared infections. If this is the case, then the effusioin must in some way be protecting the DNA from breakdown by DNases. METHODS: Here we demonstrate, using a viscosity assay, that middle ear effusions taken from children during myringotomy inhibit the breakdown of DNA in a concentration-dependent manner. Middle ear effusion homogenates 0.5-3.0 ml (1 : 10 Vol. vol/effusion: PBS) were incubated with 3 ml of DNA (0.5 mg/ml in PBS) plus 0.2 ml of DNase 1 (500 Kunitz) at 37 degrees C for 24 h. Viscosity measurements were taken at regular intervals and the changes in viscosity expressed as a percentage of time 0. DNase activity was inhibited by 1.5 ml and 3.0 ml of effusion 48% and 91%, respectively, after 30 min incubation. CONCLUSION: This study demonstrated that effusions have the ability to inhibit nuclease activity, and the reported presence of non-culturable bacteria based on DNA detection by PCR could still represent fossilized remains and not viable bacteria. The same could also be true of recent reports of bacterial mRNA.2