Inactivation Of Bacterial Cells Research Articles

Quaternized polysulfones-based blends: Surface properties and performance in life quality and environmental applications

Development of new quaternized polysulfones-based blends, which possess multifunctionality, antibacterial, and sustainability fundamental characteristics, was performed as a result of membrane technology requirements. Therefore, solutions of ionic polysulfones (PSFQ) with a tunable content of cellulose acetate phthalate (CAP) have been processed to obtain new membrane materials explored for wastewater treatment. The cumulative effects generated by the CAP versatile nature along with molecular functionalization of polysulfone were responsible for improving the workability, surface properties, and membranes performance, minimizing the adhesion of organic foulants/bacteria and maximizing the inactivation of bacterial cells. Additionally, the specific microarchitecture, determined using atomic force microscopy, represents excellent scaffolds as porous membranes. By evaluation of bacterial adhesion to surface, the practical applicability of membranes was tested, in order to clarify their suitability for life quality and environmental applications. The performed experiments represent the basis to future applications of these blends as membranes and confirm their performance in industrial field.

Mechanistic insight into the in vitro toxicity of graphene oxide against biofilm forming bacteria using laser-induced breakdown spectroscopy.

While the cytotoxicity of graphene oxide (GO) has been well established, its bactericidal mechanism, however, has yet to be elucidated to advance GO-based biomedical and environmental applications. In an attempt to better understand the bactericidal action of GO, herein we studied the interactions of GO with Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus cells using physical techniques and chemical probes, respectively. In particular, a novel laser-induced breakdown spectroscopy (LIBS) based elemental fingerprint analysis revealed notable differences between viable and non-viable cells based on the difference in the concentration of trace inorganic elements in complex bacterial systems, which reflect cellular membrane integrity. Lower emission intensities from essential inorganic ions in the GO-treated cells offered explicit evidence on the efflux of intracellular molecules from the bacteria through damaged cell membranes. Furthermore, a detailed structural and morphological investigation of bacterial membrane integrity confirmed GO-induced membrane stress upon direct contact interactions with bacterial cells, resulting in the disruption of cellular membranes. Moreover, the generation of intracellular reactive oxygen species (ROS) in the presence of an added antioxidant underlined the role of GO-mediated oxidative stress in bacterial cell inactivation. Thus, by correlating the changes in the bacterial elemental compositions with the severe morphological alterations and the high ROS production witnessed herein, we propose that the bactericidal mechanism of GO is likely to be the synergy between membrane and oxidative stress towards both tested species. Our findings offer useful guidelines for the future development of GO-based antibacterial surfaces and coatings.

Removal of Heavy Metal Ions From Aqueous Solutions Using Bacillus subtilis Biomass Pre‐Treated by Supercritical Carbon Dioxide

Surface properties of bacterial cells enable them to be used in the removal of heavy metals. Moreover, the inactivation of bacterial cells via chemical or physical processes may enhance the biosorption efficiency for heavy metals. The use of supercritical carbon dioxide (SC‐CO2) to inactivate bacteria based on destruction of cell walls took place due to the pressurized CO2. In this study, the pre‐treatment of Bacillus subtilis cells by SC‐CO2 (BM1), steam autoclaving (BM2), and untreated living cells (BM3) were investigated to show improvement in heavy metal removal. The removal process was optimized based on the main factors affecting bacterial biosorption, which included heavy metals ion concentrations, cell biomass concentrations, pH, time, and temperature. The efficiency of bacterial biomass in removing Ni2+ ions in the presence of different concentrations of Pb2+, Cu2+, Zn2+, and Cd2+ was also tested. BM1 and BM2 exhibited the highest potential for the removal of nickel ions in comparison to BM3. The maximum efficiency was 98.54, 99.2, and 96.3% for BM1, BM2, and BM3, respectively. Moreover, BM1 displayed a higher biosorption capacity for Ni2+, Cu2+, Zn2+, Pb2+, and Cd2+ (>150 mg g−1) than BM2 and BM3. Biosorption on bacterial cell biomass of Ni2+ ions shows lower removal affinity in the presence of other metal ions. In conclusion, the pre‐treatment of bacterial cells biomass by SC‐CO2 enhanced the removal process of heavy metal ions compared to untreated living cells.

Inactivation of pathogenic bacteria in food matrices: high pressure processing, photodynamic inactivation and pressure-assisted photodynamic inactivation

Traditional food processing methods frequently depend on the application of high temperature. However, heat may cause undesirable changes in food properties and often has a negative impact on nutritional value and organoleptic characteristics. Therefore, reducing the microbial load without compromising the desirable properties of food products is still a technological challenge. High-pressure processing (HPP) can be classified as a cold pasteurization technique, since it is a non-thermal food preservation method that uses hydrostatic pressure to inactivate spoilage microorganisms. At the same time, it increases shelf life and retains the original features of food. Photodynamic inactivation (PDI) is also regarded as promising approach for the decontamination of food matrices. In this case, the inactivation of bacterial cells is achieved by the cytotoxic effects of reactive oxygens species (ROS) produced from the combined interaction of a photosensitizer molecule, light and oxygen. This short review examines some recent developments on the application of HPP and PDI with food-grade photosensitizers for the inactivation of listeriae, taken as a food pathogen model. The results of a proof-of-concept trial of the use of high-pressure as a coadjutant to increase the efficiency of photodynamic inactivation of bacterial endospores is also addressed.

Comment on "Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia coli".

The recent discovery of nanostructured surfaces induced rupturing of bacterial cells has seen a surge in the development of surfaces for the physical killing of bacteria towards the fight against bacterial colonisation of abiotic surfaces. It is established that the nanoarchitecture is directly responsible for the inactivation of bacterial cells; however the mechanobactericidal action remains to be fully elucidated. Here we report the fabrication of superhydrophilic and superhydrophobic black silicon surfaces with well-defined surface geometries and wettability which are responsible for inactivating approximately 98% of P. aeruginosa cells and 97% of S. aureus cells. Increased adhesion of bacterial cells onto superhydrophobic surfaces was not accompanied by increased secretion of extracellular polymeric substances. Surface hydrophobicity has been demonstrated to be statistically unimportant in determining the bactericidal efficiency of nanostructured surfaces.

Combination of endolysins and high pressure to inactivate Listeria monocytogenes

Outbreaks of listeriosis are often related to the consumption of low-processed ready-to-eat food products (e.g. soft cheeses or smoked fish) contaminated with Listeria monocytogenes. Traditional preservation techniques, such as heat treatment, cannot eliminate Listeria from these products without strongly affecting the quality of the foods. We therefore investigated the use of endolysin (PlyP40, Ply511, or PlyP825) in combination with high hydrostatic pressure processing to kill L. monocytogenes in buffer. The results demonstrated a more than additive effect when both treatments were combined. For example, whereas 0.16 μg/mL PlyP825 or 300 MPa (1 min, 30 °C) applied individually reduced the cell count by 0.2 and 0.3 log cfu, respectively, a combined treatment resulted in a reduction of 5.5 log cfu. Similar results were obtained for the other endolysins combined with high pressure processing. We also showed that the synergistic inactivation of cells by endolysin and HHP is possible at a pressure level of only 200 MPa (2 min, 30 °C). Thus, the application of endolysins did not only substantially increase the bactericidal effect of high pressure, but it also enabled the inactivation of bacterial cells at much lower pressure levels. This shows the potential of using such combined processes for the inactivation of L. monocytogenes and food preservation.

Influence of nanoscale topology on bactericidal efficiency of black silicon surfaces

The nanostructuring of materials to create bactericidal and antibiofouling surfaces presents an exciting alternative to common methods of preventing bacterial adhesion. The fabrication of synthetic bactericidal surfaces has been inspired by the anti-wetting and anti-biofouling properties of insect wings, and other topologies found in nature. Black silicon is one such synthetic surfaces which has established bactericidal properties. In this study we show that time-dependent plasma etching of silicon wafers using 15, 30, and 45 min etching intervals, is able to produce different surface geometries with linearly increasing heights of approximately 280, 430, and 610 nm, respectively. After incubation on these surfaces with Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacterial cells it was established that smaller, more densely packed pillars exhibited the greatest bactericidal activity with 85% and 89% inactivation of bacterial cells, respectively. The decrease in the pillar heights, pillar cap diameter and inter-pillar spacing corresponded to a subsequent decrease in the number of attached cells for both bacterial species.

Chloride ion-driven transformation from Ag3PO4 to AgCl on the hydroxyapatite support and its dual antibacterial effect against Escherichia coli under visible light irradiation.

Visible light-driven photocatalytic inactivation of Escherichia coli was performed using hydroxyapatite-supported Ag3PO4 nanocomposites (Ag3PO4/HA). The antibacterial performance was evaluated by the methods of zone of inhibition plates and minimum inhibitory concentration test. X-ray diffraction (XRD) and transmission electron microscopy (TEM) were employed to investigate the instability and transformation of the nanocomposite by comparing the crystalline, phase, and the morphology before and after exposure to Luria-Bertani culture medium under visible light irradiation. Ag3PO4 nanoparticles on the support were found to be shortly transformed into AgCl due to high chloride concentration of Luria-Bertani culture medium. The AgCl/HA nanocomposite showed both excellent intrinsic antibacterial performance contributed by the released silver ions and visible light-induced photocatalytic disinfection toward E. coli cells. This dual antibacterial function mechanism was validated by trapping the hydroxyl free radical and detecting the silver ions during the photocatalytic antibacterial process. The morphological change of E. coli cells in different reaction intervals was obtained by scanning electron microscopy (SEM) to complementally verify photocatalytic inactivation of E. coli. This work suggests that an essential comparison study is required for the antibacterial materials before and after the photocatalytic inactivation of bacterial cells using Ag3PO4 nanoparticles or Ag3PO4-related nanocomposites in mediums containing high-concentration chloride ions.

Photodynamic inactivation of antibiotic-resistant bacteria and biofilms by hematoporphyrin monomethyl ether.

The worldwide increase in bacterial antibiotic resistance has led to a search for alternative antibacterial therapies. A promising approach to killing antibiotic-resistant bacteria is photodynamic antimicrobial chemotherapy, which uses light in combination with a photosensitizer to induce a phototoxic reaction. We evaluated the photodynamic inactivation (PDI) efficiency of hematoporphyrin monomethyl ether (HMME) on antibiotic-resistant bacteria and biofilms. HMME exhibited no significant dark toxicity and provided dose-dependent inactivation of antibiotic-resistant bacteria and biofilms. After incubation with 100-μM HMME and irradiation with 72-J cm(-2) white light, 4.19-7.59 log10 reductions in survival were achieved in planktonic suspension. Antibiotic-resistant strains were as susceptible to PDI in biofilms as in planktonic suspensions, but the inactivation of bacterial cells in biofilms was attenuated. In addition, gram-positive bacterial strains and biofilms were more susceptible than gram-negative strains and biofilms to the PDI effect of HMME. Thus, HMME is a promising photosensitizer for the treatment of infectious diseases caused by antibiotic-resistant bacteria, especially gram-positive bacteria.

Photocatalytic inactivation of Escherichia coli—The roles of genes in β-oxidation of fatty acid degradation

A genetic study on the relationship between cell membrane fatty acid profile and photocatalytic inactivation efficiency of Escherichia coli was conducted. Photocatalytic inactivation of a parental strain (E. coli BW25113) and its seven isogenic mutants with deletion of single gene involving in β-oxidation of fatty acid degradation (fad) were compared. Most of the mutants involved in fatty acid degradation did not show significant difference in susceptibility towards photocatalytic inactivation compared with the parental strain, except that E. coli JW1176 (fadR mutant) and E. coli JW3935 (fabR mutant) showed a lower and higher sensitivity than the parental strain, respectively. Fluorescence microscopic analysis showed that the loss of cell permeability preceded the inactivation of bacterial cells. The results of temperature pre-treatment and fatty acid profiles of the parental strain and fadR mutant indicated that the alteration in cell membrane fatty acid composition played an important role, but not the most crucial one to affect the susceptibility of bacterial cell towards photocatalytic inactivation. The results in this study demonstrated the importance of cell membrane in the bacterial defense system against photocatalytic inactivation.

The role and synergistic effect of the light irradiation and H2O2 in photocatalytic inactivation of Escherichia coli

Inactivation of Escherichia coli K-12 was conducted by applying a continuous supplying of commercial H2O2 to mimic the H2O2 production in a photocatalytic system, and the contribution of H2O2 in photocatalytic inactivation was investigated using a modified “partition system” and five E. coli mutants. The concentration of exogenous H2O2 required for complete inactivation of bacterial cells was much higher than that produced in-situ in common photocatalytic system, indicating that H2O2 alone plays a minor role in photocatalytic inactivation. However, the concentration of exogenously produced H2O2 required for effective inactivation of E. coli K-12 was much lower when the light irradiation was applied. To further investigate the possible physiological changes, inactivation of E. coli BW25113 (the parental strain), and its corresponding isogenic single-gene deletion mutants with light pretreatment was compared. The results indicate that light irradiation increases the bacterial intracellular Fe2+ level and favors hydroxyl radical (OH) production via the catalytic reaction of Fe2+, leading to increase in DNA damage. Moreover, the results indicate that the properties of light source, such as intensity and major emission wavelength, may alter the physiology of bacterial cells and affect the susceptibility to in-situ resultant H2O2 in the photocatalytic inactivation processes, leading to significant influence on the photocatalytic inactivation efficiencies of E. coli K-12.

Interaction of Graphene Oxide with Bacterial Cell Membranes: Insights from Force Spectroscopy

Understanding the interactions of graphene oxide (GO) with biological membranes is crucial for the evaluation of GO’s health and environmental impacts, its bactericidal activity, and to advance graphene-based biological and environmental applications. In an effort to understand graphene-induced bacterial inactivation, we studied the interaction of GO with bacterial (Escherichia coli) cell membranes using atomic force microscopy (AFM). Toward this goal, we devised a polydopamine-assisted experimental protocol to functionalize an AFM probe with GO nanosheets, and used AFM-based force spectroscopy to measure cell membrane–GO interaction forces. Our results show that GO–cell interactions are predominantly repulsive, with only sporadic adhesion forces being measured upon probe pull-off, which we attribute to lipopolysaccharide bridging. We provide evidence of the acellular oxidation of glutathione by GO, underscoring the role of oxidative pathways in GO-mediated bacterial cell inactivation. Our force spectrosc...

Selective photodynamic inactivation of bacterial cells over mammalian cells by new triarylmethanes.

Three new triarylmethane dyes (TAMs), MPCV, DPCV, and AEV, were synthesized and their photodynamic inactivation abilities against E. coli and human pulmonary carcinoma A549 cells were compared to two commercial TAMs, CV and EV. The enhanced hydrophilicity of MPCV and AEV decreases their cellular uptake to A549 cells dramatically. However, their binding affinity toward E. coli cells are comparable to that of CV and EV by virtue of the improved electrostatic attraction with highly negatively charged E. coli outer membranes. MPCV and AEV were also found to generate hydroxyl radicals more efficiently upon irradiation than CV and EV. Consequently, MPCV and AEV exhibited markedly improved photodynamic inactivation of E. coli cells but remarkably diminished photodynamic inactivation of A549 cells than CV and EV. The photodynamic inactivation ability of DPCV was much lower than that of CV due to its high propensity for bleaching in neutral aqueous solution. Our work demonstrates that the introduction of protonatable groups in a proper manner into the structures of TAMs may lead to selective binding and photodynamic inactivation toward bacterial cells over mammalian cells. This strategy may be extended to other types of photodynamic antimicrobial chemotherapy (PACT) agents to improve their clinical potential.

Systematic approach to in-depth understanding of photoelectrocatalytic bacterial inactivation mechanisms by tracking the decomposed building blocks.

A systematic approach was developed to understand, in-depth, the mechanisms involved during the inactivation of bacterial cells using photoelectrocatalytic (PEC) processes with Escherichia coli K-12 as the model microorganism. The bacterial cells were found to be inactivated and decomposed primarily due to attack from photogenerated H2O2. Extracellular reactive oxygen species (ROSs), such as H2O2, may penetrate into the bacterial cell and cause dramatically elevated intracellular ROSs levels, which would overwhelm the antioxidative capacity of bacterial protective enzymes such as superoxide dismutase and catalase. The activities of these two enzymes were found to decrease due to the ROSs attacks during PEC inactivation. Bacterial cell wall damage was then observed, including loss of cell membrane integrity and increased permeability, followed by the decomposition of cell envelope (demonstrated by scanning electronic microscope images). One of the bacterial building blocks, protein, was found to be oxidatively damaged due to the ROSs attacks, as well. Leakage of cytoplasm and biomolecules (bacterial building blocks such as proteins and nucleic acids) were evident during prolonged PEC inactivation process. The leaked cytoplasmic substances and cell debris could be further degraded and, ultimately, mineralized with prolonged PEC treatment.

A novel bioadhesive semisolid formulation containing chitosan and tetracycline/layered clay complexes for local delivery into periodontal pocket

An intrapocket non-conventional bioadhesive semisolid formulation based on tetracycline layered clay complexes and chitosan was developed to ensure extended drug release properties and adequate permanence time at the site of action. A Dohelert experimental design was used to discriminate among seven possible formulations, using viscosity as response variable. The formulation containing chitosan 2% (w/w) and complex (15% w/w) was selected as better candidate and was characterised for rheological behaviour, bioadhesive properties, microbicidal effect and drug delivery features. The formulation maintained the antibacterial activity of the drug against Streptococcus vestibularis, reaching a complete inactivation of bacterial cells after 24 h. The presence of the complex in the formulation not only did not induce any change in the intrinsic bioadhesive properties of chitosan, ensuring a good permanence time in the site of application but also resulted in drug release/wash away profiles compatible with a once a week administration of tetracycline HCl into the periodontal pocket.

Effect of pulsed light on structural and physiological properties of Listeria innocua and Escherichia coli

The application of broad-spectrum intense light pulses is an innovative nonthermal technology for the decontamination of packaging materials, liquids or foodstuffs. The objective of this study was the fundamental investigation of the cellular impact of a pulsed light treatment on Listeria innocua and Escherichia coli. Flow cytometry in combination with different fluorescent stains, conventional plate count technique and a viability assay were applied to investigate the effects of a pulsed light treatment on the physiological properties of L.innocua and E.coli. The results showed that loss of cultivability occurred at considerably lower fluences than the shutdown of cellular functions such as the depolarization of cell membranes, the loss of metabolic, esterase and pump activities or the occurrence of membrane damage. Therefore, a considerable proportion of cells appeared to have entered the viable but nonculturable (VBNC) state after the pulsed light treatment. A high percentage of L.innocua was able to maintain certain cellular vitality functions after storage overnight, whereas a further decrease in vitality was observed in case of E.coli. The loss of culturability was on the other hand directly accompanied by the formation of reactive oxygen species (ROS) and DNA damages, which were assessed by the ROS-sensitive probe DCFH-DA and RAPD-PCR, respectively. A significant discrepancy between conventional plate counts and different viability staining parameters was observed, which shows that a pulsed light treatment does not cause an immediate shutdown of vitality functions even when the number of colony-forming units already decreased for more than 6 log10 sample(-1) . Oxidative stress with concomitant damage to the DNA molecule showed to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes. The presented results suggest an UV light-induced photochemical rather than a photothermal or photophysical inactivation of bacterial cells by pulsed light under the applied experimental conditions. Flow cytometry in combination with different viability stains proved to be a suitable technique to gain deeper insight into the cellular response of bacteria to inactivation processes like a pulsed light treatment.

Inactivation of Lactococcus lactis ssp. cremoris cells in a droplet during convective drying

Spray drying is a less costly alternative to freeze drying in the mass-production of active dry microorganisms, if the drying conditions could be optimized to preserve cell viability. As spray drying is akin to a black-box process, in this study we used an alternative approach of a single droplet drying to study how drying conditions affect the inactivation of bacterial cells. The inactivation histories of Lactococcus lactis ssp. cremoris were investigated at air temperatures of 70, 90, and 110°C. It was found that the viability of L. cremoris cells could be maintained at approximately the original level for extended drying durations (60–210s), despite the high air temperatures. When plotted against droplet temperature Td, the inactivation rate kd at six drying conditions formed a general trend. An inactivation model was proposed to describe different inactivation histories under varied drying conditions. The description closely followed the experimental data, reported for the first time in literature. kd increased rapidly after Td passed a transition temperature range of 50–65°C, coinciding with the onset temperature for denaturation of bacterial ribosomes. Other environmental parameters affecting inactivation are discussed to better understand the integrated effects of multiple stresses experienced by bacterial cells during convective drying.

Combination of Microsecond and Nanosecond Pulsed Electric Field Treatments for Inactivation of Escherichia coli in Water Samples

Inactivation of microorganisms with pulsed electric fields is one of the nonthermal methods most commonly used in biotechnological applications such as liquid food pasteurization and water treatment. In this study, the effects of microsecond and nanosecond pulses on inactivation of Escherichia coli in distilled water were investigated. Bacterial colonies were counted on agar plates, and the count was expressed as colony-forming units per milliliter of bacterial suspension. Inactivation of bacterial cells was shown as the reduction of colony-forming units per milliliter of treated samples compared to untreated control. According to our results, when using microsecond pulses the level of inactivation increases with application of more intense electric field strengths and with number of pulses delivered. Almost 2-log reductions in bacterial counts were achieved at a field strength of 30 kV/cm with eight pulses and a 4.5-log reduction was observed at the same field strength using 48 pulses. Extending the duration of microsecond pulses from 100 to 250 μs showed no improvement in inactivation. Nanosecond pulses alone did not have any detectable effect on inactivation of E. coli regardless of the treatment time, but a significant 3-log reduction was achieved in combination with microsecond pulses.

Bactericidal efficiency and mode of action: A comparative study of photochemistry and photocatalysis

In order to compare the disinfection potential of photocatalysis and photochemistry, the effects of these two processes on bacteria in water were investigated under exposure to UV-A and UV-C. The well-known bacterial model Escherichia coli (E. coli) was used as the experimental organism. Radiation exposure was produced with an HPK 125 W lamp and the standard TiO2 Degussa P-25 was used as the photocatalyst.Firstly, the impact of photocatalysis and photochemistry on the cultivability of bacterial cells was investigated. UV-A radiation resulted in low deleterious effects on bacterial cultivability but generated colonies of size smaller than average. UV-C photocatalysis demonstrated a greater efficiency than UV-A photocatalysis in altering bacterial cultivability. From a cultivability point of view only, UV-C radiation appeared to be the most deleterious treatment.A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit was then used to assess the modifications in bacterial membrane permeability. UV-A radiation did not induce any alterations in bacterial permeability for 420 min of exposure whereas only a few minutes of exposure to UV-C radiation, with the same total radiance intensity, induced total loss of permeability. Moreover, after 20 and 60 min of exposure to UV-C and UV-A photocatalysis respectively, all bacteria lost their membrane integrity, suggesting that the bacterial envelope is the primary target of reactive oxygen species (ROS) generated at the surface of TiO2 photocatalyst. These results were further confirmed by the formation of malondialdehyde (MDA) during the photocatalytic inactivation of bacterial cells and suggest that destruction of the cell envelope is a key step in the bactericidal action of photocatalysis. The oxidation of bacterial membrane lipids was also correlated with the monitoring of carboxylic acids, which can be considered as representatives of lipid peroxidation by-products.Finally, damages to bacterial morphology induced by UV-C photocatalysis and photochemistry were investigated through Scanning electron microscopy (SEM). Bacterial cells were observed on microscopy pictures at exposure durations corresponding to a loss of cultivability. After 90 min of exposure to UV-C radiation, bacterial cells showed little alteration of their outer membrane whereas they suffered deep deleterious damages under UV-C photocatalysis exposure.

Effects of High Hydrostatic Pressure on Bacterial Growth on Human Ossicles Explanted from Cholesteatoma Patients

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.