Objectives Antimicrobials such as chlorine dioxide, peracetic acid and hydrogen peroxide (H2O2) share a basic mechanism of action (chemical oxidation of cellular components), but profound differences ...arise in their efficacy against microorganisms. Optimization of activity requires an understanding of their interaction with microbial targets and a clear differentiation between the chemical efficacies of each oxidative biocide. This study aimed to elucidate the biochemical mechanisms of action of oxidizing biocides at a macromolecular level, using amino acids, protein and an enzyme as model substrates for the action of each biocide. Methods The interactions of a number of oxidising agents (liquid and gaseous H2O2, ClO2, peracetic acid formulations) with amino acids, proteins (bovine serum albumin and aldolase) and enzymes were investigated by spectrophotometry, SDS-PAGE and alkaline phosphatase activity measurements. Results Biocide reactions yielded different types of oxidative structural change and different degrees of oxidation to amino acids and proteins, and differences in activity against a microbial enzyme. In particular there was a marked difference in the interactions of liquid H2O2 and gaseous H2O2 with the macromolecules, the latter causing greater oxidation; these results explain the dramatic differences in antimicrobial efficacy between liquid and gas peroxide. Conclusions These results provide a comprehensive understanding of the differences in interactions between a number of oxidizing agents and macromolecules commonly found in microbial cells. Biochemical mechanistic differences between these oxidative biocides do exist and lead to differential effects on macromolecules. This in turn could provide an explanation as to their differences in biocidal activity, particularly between liquid and gas peroxide.
The aim of this study was to determine the stability and purity of a phage cocktail to be delivered by nebulization. A cocktail of three phages active against Pseudomonas aeruginosa isolates from ...cystic fibrosis patients was developed for a potential nebulized formulation. The individual phages were examined for their retention of activity over time, while the phage cocktail was analysed for bacterial contaminant and endotoxin level according to regulatory requirements for nebulized products. The phage cocktail was nebulized using a Porta‐neb nebulizer connected to an Anderson cascade impactor. The three phages retained activity over a period of 180 days storage at room temperature and at 4°C. Nebulized phages were recovered in the lower stages of the cascade impactor indicative of potential delivery deep into the lungs. The phage cocktail met bacterial limits but the endotoxin levels measured with the Limulus amoebocyte lysate (LAL) test remained considerably in excess of acceptable levels even following purification. These findings suggest that nebulization of phage is a viable delivery option; although, there is a need for appropriate depyrogenation strategies to remove bacterial endotoxins from phage‐based preparations to meet regulatory requirements. SIGNIFICANCE AND IMPACT OF THE STUDY: With increasing reports of bacterial resistance to antibiotics and the lack of new antibiotics being produced, bacteriophage therapy is becoming an attractive alternative. There has been no published report on the quality assurance of bacteriophage product to date. This is the first study on the quality assurance of a Pseudomonas aeruginosa phage cocktail following pharmacopoeial requirements. The presence of bacterial endotoxin was found to be the key stumbling block for meeting regulatory criteria.
Summary Background Antimicrobial surfaces aim to reduce microbial bioburden and improve hygiene. The current antimicrobial surface efficacy test (ISO22196) is an initial screening test but its ...conditions, high temperature (37 °C) and relative humidity (RH) (100%) bear little relationship to in-use conditions. Aim To develop an antimicrobial surface efficacy test providing a realistic second-tier test, simulating in-use conditions. Methods Surface relative humidity, temperature and soiling were measured over one year at a UK hospital, enabling realistic parameters to be set for our surface efficacy test. A nebulizer, connected to a cascade impactor, aerosolized and uniformly deposited a Staphylococcus aureus suspension over test copper alloys and control stainless steel surfaces. Bacteria were enumerated following nebulization, and after a range of contact times, under 20 °C, 50% RH and 20 °C, 40% RH parameters reflecting in-use conditions; 37 °C, 100% RH was employed to reflect conditions used in ISO22196. Findings All copper alloys produced a >4 log10 reduction after 24 h under all conditions tested. Copper alloys were more effective at 37 °C, 100% RH showing a >4 log10 reduction after 30 min than at in-use conditions 20 °C, 50% RH and 20 °C, 40% RH, for which 60 min was required to achieve the same level of kill, for most but not all alloys. Conclusion The use of the nebulizer to deposit bacterial inocula on surfaces showed little variability in results. Our method was more discriminatory than the ISO22196 enabling distinction between the bactericidal surface activity, which allows for a more rigorous selection of antimicrobial surfaces for potential use in healthcare settings.
The introduction of antimicrobial surfaces into healthcare environments is believed to impact positively on the rate of healthcare-associated infections by significantly decreasing pathogen presence ...on surfaces.
To report on a novel efficacy test that uses a dry bacterial inoculum to measure the microbicidal efficacy of antimicrobial surfaces.
An aerosolized dry inoculum of Staphylococcus aureus or Acinetobacter baumannii was deposited on copper alloy surfaces or a hospital-grade stainless-steel surface. Surviving bacteria were enumerated following incubation of the inoculated surfaces at an environmentally relevant temperature and relative humidity. Damage caused to bacteria by the aerosolization process and by the different surfaces was investigated.
Dry inoculum testing showed a <2-log10 reduction in S. aureus or A. baumannii on the copper alloy surfaces tested after 24 h at 20°C and 40% relative humidity. Potential mechanisms of action included membrane damage, DNA damage and arrested cellular respiration. The aerosolization process caused some damage to bacterial cells. Once this effect was taken into account, the antimicrobial activity of copper surfaces was evident.
Our test provided a realistic deposition of a bacterial inoculum to a surface and, as such, a realistic protocol to assess the efficacy of dry antimicrobial environmental surfaces in vitro.
AIMS: Oxidizing agents such as chlorine dioxide are widely used microbicides, including for disinfection of medical equipment. We isolated a Bacillus subtilis isolate from a washer‐disinfector whose ...vegetative form demonstrated unique resistance to chlorine dioxide (0·03%) and hydrogen peroxide (7·5%). The aim of this study was to understand the mechanisms of resistance expressed by this isolate. METHODS AND RESULTS: A range of resistance mechanisms were investigated in the B. subtilis isolate and a reference B. subtilis strain (ATCC 6051) to include bacterial cell aggregation, the presence of profuse exopolysaccharide (EPS), and the expression of detoxification enzymes. The basis of resistance of the isolate to high concentrations of oxidizing agents was not linked to the presence of endospores. Although, the presence of EPS, aggregation and expression of detoxification enzymes may play a role in bacterial survival to low concentrations of chlorine dioxide, it is unlikely that the mechanisms helped tested to survive the bactericidal effect of higher oxidizer concentrations. CONCLUSIONS: Overall, the mechanisms conferring resistance to chlorine dioxide and hydrogen peroxide remains elusive. Based on recent advances in the mode of action of oxidizing agents and notably hydrogen peroxide, we postulate that additional efficient intracellular mechanisms may be involved to explain significant resistance to in‐use concentrations of commonly used high‐level disinfectants. SIGNIFICANCE AND IMPACT OF STUDY: The isolation of a highly resistant vegetative Gram‐positive bacterium to a highly reactive oxidizing agent is worrying. Understanding the mechanisms conferring such resistance is essential to effectively control such bacterial isolates. Here, we postulate that there are still mechanisms of bacterial resistance that have not been fully characterized.
To elucidate the mechanisms of spore resistance to and killing by the oxidizing microbicide peracetic acid (PAA).
Mutants of Bacillus subtilis lacking specific spore structures were used to identify ...resistance properties in spores and to understand the mechanism of action of PAA. We also assessed the effect of PAA treatment on a number of spore properties including heat tolerance, membrane integrity and germination.
The spore coat is essential for spore PAA resistance as spores with defective coats were greatly sensitized to PAA treatment. Small acid-soluble spore proteins apparently provide no protection against PAA. Defects in spore germination, specifically in germination via the GerB and GerK but not the GerA germination receptors, as well as leakage of internal components suggest that PAA is active at the spore inner membrane. It is therefore likely that the inner membrane is the major site of PAA's sporicidal activity.
PAA treatment targets the spore membrane, with some of its activity directed specifically against the GerB and GerK germination receptors.
Aims: To demonstrate that the nonlinear concentration‐dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA.
...Methods and Results: A model used successfully to describe the concentration‐dependent inhibition of bacterial growth caused by many antimicrobials was unable to describe the inhibition of P. aeruginosa by EDTA. Examination of the inhibition profiles for EDTA against P. aeruginosa revealed a biphasic inhibitory pattern suggesting different mechanisms of action at different concentrations. A modelled, two‐stage inhibitory process was shown to fit the observations. This model was then used to examine the effect of combining EDTA with other antimicrobials. The apparent synergy of mixtures of EDTA with quaternary ammonium surfactants (QAC) and specific antibiotics was successfully modelled. Minimum inhibitory concentrations (MIC) of the QAC and that of oxacillin and cefamandole were reduced by a factor of 3–10, whereas ampicillin was reduced by a factor of 70 from an MIC of 1524 to 21 mg l−1 in the presence of 500 mg l−1 of EDTA.
Conclusions: A nonlinear concentration‐dependent inhibition of P. aeruginosa by EDTA gives rise to apparent observation of synergy with other antimicrobials.
Significance and Impact of the Study: This is a further example where the current methodology for the examination of antimicrobial synergy (the summed fractional inhibitory concentrations) leads to false conclusions.
This study reports the activity of two biguanides against MS2 bacteriophage used as a surrogate virus for nonenveloped mammalian viruses and provides an explanation as to their apparent limited ...efficacy. When tested in a standard suspension test, two polyhexamethylene biguanides (PHMB), VANTOCILtrade mark sign TG and COSMOCILtrade mark sign CQ, reduced the viability of MS2 by only 1-2 log₁₀ PFU ml⁻¹. Exposure time up to 30 min did not affect the activity of the biguanides, although both PHMB were shown to strongly interact with MS2 proteins. Inactivation kinetics and change in virus hydrophobicity suggested that PHMB induces the formation of viral aggregates. This hypothesis was supported using dynamic light scattering that showed an increase in viral aggregates sizes (up to 500 nm) in a concentration-dependent manner. It has been reported that viral aggregation is responsible for virus survival to the biocide exposure. Here, this might be the case, because the virucidal activity of the biguanides was modest and viral aggregation important. The formation of viral aggregates during virus exposure to PHMB was unlikely to overestimate the virucidal potential of the biguanides.
Ocular biomaterials and implants Lloyd, Andrew W.; Faragher, Richard G.A.; Denyer, Stephen P.
Biomaterials,
04/2001, Letnik:
22, Številka:
8
Journal Article
Recenzirano
The maintenance of vision is a key determinant of healthy ageing. This has been facilitated over recent decades by the development of a wide range of implants and biomedical devices to correct the ...functional deficiencies of disease, age and ocular trauma. This brief overview provides an insight into the structure of this unique organ, the major physiological functions of the component tissues and the present state of the art with respect to modern ocular implants. The review focuses primarily on the existing limitations of existing ocular biomaterials used in the fabrication of contact lenses, intraocular lenses, glaucoma filtration implants, keratoprostheses, intracorneal implants, scleral buckles and viscoelastic replacement agents. The challenge of improving ocular compatibility and ensuring the longevity of indwelling ocular devices is addressed along with the need to improve the physicochemical and mechanical properties of existing ocular biomaterials.
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