Abstract
BackgroundHigh hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates.MethodologyTwenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium.Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy.Principal FindingsA variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms.High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.
Highlights
Cholesteatoma is defined as the presence of keratinizing squamous epithelium in the tympanic cavity
The present study is the first to assess the effects of High hydrostatic pressure (HHP) treatment on microorganisms colonizing human ossicles that were obtained from cholesteatoma patients
The study had several objectives: i) to characterize typical microbes colonizing the ossicles of cholesteatoma patients; ii) to assess whether HHP treatment can effectively remove colonizing microbes at settings previously shown to eradicate cholesteatoma cell growth on human ossicles without harming the ossicle itself [19]; iii) to investigate whether a combination of HHP and antibiotics increases the effects of treatment on colonizing microbes, and iv) to determine the potential influence of biofilm organization on microbial survival during exposure to HHP treatment
Summary
Cholesteatoma is defined as the presence of keratinizing squamous epithelium in the tympanic cavity. Among the factors discussed are molecular dysregulation of keratinocytes and external stimulation by pro-inflammatory cytokines, growth factors and/or bacterial toxins. Inflammatory mediators such as histamine and platelet-activating factor (PAF) appear to be involved in disease progression since they can cause eustachian tube dysfunction resulting in decreased mucociliary clearance. Another possible mechanism is that inflammatory mediators such as tumor necrosis factor alpha (TNF-a), interleukin 1 (IL-1), and PAF induce mucin secretion in the middle ear epithelium. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates
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