Bacillus subtilis vegetative isolate surviving chlorine dioxide exposure: an elusive mechanism of resistance.

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.

Stability and purity of a bacteriophage cocktail preparation for nebulizer delivery.

UNLABELLED: 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.

Bacterial spore structures and their protective role in biocide resistance.

The structure and chemical composition of bacterial spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main spore structures, highlighting their relevance to spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future.

Rapid and quantitative automated measurement of bacteriophage activity against cystic fibrosis isolates of Pseudomonas aeruginosa.

INTRODUCTION: Pseudomonas aeruginosa is an opportunistic pathogen and is the main cause of respiratory infection in cystic fibrosis patients. Most strains prevalent within the UK are resistant to two or more antibiotics leading to the search for new therapeutic strategies including the use of bacteriophages. METHODS AND RESULTS: The infectivity of four bacteriophages was increased using an enhancement protocol based on the use of pomegranate rind extract. Their efficacy against 14 Ps. aeruginosa strains was measured using a qualitative streak test and a novel quantitative assay based on the Bioscreen C microbial growth analyzer. Streak test analysis illustrated an increase in the lytic activity of enhanced bacteriophages, whereas Bioscreen analysis showed that both enhanced and unenhanced bacteriophages failed to meet acceptable levels of activity in c. 50% of strains tested. CONCLUSIONS: The quantitative Bioscreen C analyzer showed comparable but not identical results in phage activity and identified significant bacterial re-growth by 20 h postinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: With the resurgence of interest in bacteriophage therapy against infectious bacterial diseases, a rapid high throughput quantitative method for screening phage activity and bacterial resistance is required. The use of the Bioscreen C analyzer meets these criteria and was shown to be more stringent than the traditional streak test.

Polyhexamethylene biguanide exposure leads to viral aggregation.

AIMS: 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. METHODS AND RESULTS: When tested in a standard suspension test, two polyhexamethylene biguanides (PHMB), VANTOCIL TG and COSMOCIL CQ, reduced the viability of MS2 by only 1-2 log(10) 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Impact of a dry inoculum deposition on the efficacy of copper-based antimicrobial surfaces.

BACKGROUND: 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. AIM: To report on a novel efficacy test that uses a dry bacterial inoculum to measure the microbicidal efficacy of antimicrobial surfaces. METHODS: 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. FINDINGS: 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. CONCLUSIONS: 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.

Control of microbial contamination of Franz diffusion cell receptor phase in the development of transcutaneous breast cancer therapeutics.

AIMS: To determine the micro-organism contamination of excised porcine (pig) ear, and evaluate the use of Cyclopore track-etched membranes (CTEM) for preventing ingress into Franz-type diffusion cells. METHODS: Swabs were taken from four locations and used to inoculate Tryptone Soya Agar (TSA) and Sabouraud Dextrose Agar (SDA) plates. Diffusion cells were assembled to include porcine skin with and without CTEM, and the receptor phase sampled periodically and spread onto plates. RESULTS: Five distinct colony types were isolated after incubation of all swabs on TSA plates at 37 degrees C; on SDA plates, one fungal colony was found at 30 degrees C and one at 37 degrees C. The SDA agar plate incubated at 30 degrees C resulted in the growth of a large diffused white fungal colony. No regional differences were observed. Without the CTEM, the receptor phase became contaminated within 6 h. With the CTEM present, microbial ingress was substantially retarded with visible presumptive fungal growth occurring at 24 h and detectable contamination on both microbiological media at 48 h. CONCLUSIONS: As expected, the native porcine ears were considerably contaminated. The ingress of contamination into the diffusion cell receptor phases can be largely, but not entirely, eliminated using CTEM. The addition of antimicrobial agents was necessary to eliminate micro-organisms that were observed at later time points. SIGNIFICANCE AND IMPACT OF THE STUDY: This article, while highlighting the presence of a high number of micro-organisms on native porcine skin, presents a practical means to reduce the risk of microbial contamination in transdermal/transcutaneous permeation studies, particularly in the study of cell cultures grown within Franz diffusion cell receptor compartments.

Resistance and cross-resistance to oxidising agents of bacterial isolates from endoscope washer disinfectors.

Bacteria isolated from washer disinfectors using chlorine dioxide as a high-level disinfectant were exposed to peracetic acid, chlorine dioxide and hydrogen peroxide to investigate their susceptibility and possible bacterial cross-resistance to these highly reactive oxidising biocides. A standard suspension test was used to establish a rate of kill of these biocides against two stable isolates (Bacillus subtilis and Micrococcus luteus). Suspension tests demonstrated that 'in use' concentrations were not always effective to provide the required disinfection efficacy within recommended exposure times and in some instances a 60min exposure was necessary to achieve a reduction in number by a factor of 10(5). It appears that vegetative Gram-positive isolates can become resistant to oxidising agents in vitro, and that cross-resistance to related compounds can occur. Since these bacteria are deemed to be susceptible to highly reactive biocides, there should be further study of the resistance mechanisms in these isolates to explain their survival.

The development of a new three-step protocol to determine the efficacy of disinfectant wipes on surfaces contaminated with Staphylococcus aureus.

We developed a three-step protocol to quantify the efficacy of disinfectant wipes, their ability to remove and prevent microbial transfer from surfaces and their overall antimicrobial activity. Meticillin-resistant (MRSA) or -susceptible (MSSA) Staphylococcus aureus (6-7 log(10)cfu) were inoculated onto stainless steel discs with or without organic load and dried. Grapefruit extract-containing test wipes and unmedicated control wipes were used. In step 1, wipes were mechanically rotated against surfaces for 10s at 60rpm, exerting a weight of 100+/-5g. Bacterial removal was assessed by transferring the steel discs to neutraliser, resuspending and counting remaining bacteria. In step 2, bacterial transfer from wipes was assessed by eight consecutive mechanical adpression transfers to agar/neutraliser plates. Step 3 was the measurement of antimicrobial activity by direct inoculation of the wipes for 10s followed by neutralisation and enumeration. Test wipes achieved a significantly higher bacterial cell removal than control wipes on all surfaces (P<0.05). The low bactericidal activity of the wipes (<1 log(10) reduction when directly inoculated) and the subsequent survival of bacteria on the wipes, however, led to repeated microbial transfer when initially high contamination levels were present. There were no differences between MRSA and MSSA in removal, transfer or antimicrobial activity. The three-step method is a useful tool for developing future guidelines to assess the ability of wipes to disinfect surfaces.

Alcohol ethoxylates mediate their bacteriostatic effect by altering the cell membrane of Escherichia coli NCTC 8196.

The minimum inhibitory concentration (MIC) of a homologous series of alcohol ethoxylates with the same head group size (E6) but differing in the number of carbon atoms in their 'tail group' from 10 to 16 was determined for Staphylococcus aureus NCTC 4163 and Escherichia coli NCTC 8196 using a turbidimetric assay. All the surfactants tested demonstrated bacteriostatic activity against both organisms. A tetrazolium assay showed that C14E6 and C16E6 had little effect on the membrane-bound dehydrogenase enzyme activity of E. coli NCTC 8196 compared with C10E6 and C12E6. C10E6 caused leakage both of K(+) and nucleotides in a concentration-dependent manner above its MIC of 0.2 mM. C12E6 caused some leakage at concentrations below its MIC (0.12 mM).

A cell kinetic analysis of human umbilical vein endothelial cells.

Cultures of normal human cells 'age' and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43+/-0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47+/-0.3% as assessed by senescence associated beta-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31+/-0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.

Control of staphylococcal adhesion to polystyrene surfaces by polymer surface modification with surfactants.

The adherence of three clinical isolates of Staphylococcus epidermidis to model polystyrene surfaces was studied in vitro using epifluorescent image analysis. A series of 16 Pluronic surfactants (A-B-A block copolymers where A is poly(ethylene oxide) (PEO) and B is poly(propylene oxide) (PPO)) were used as surface modifiers for the model polystyrene surfaces. Substantial reductions (up to 97%) in bacterial adhesion levels were achieved with all copolymers tested, irrespective of the PPO or PEO block lengths. It appears likely that such treatments create a sterically stabilized surface with adsorbed PEO chains, conferring nonspecific anti-adhesive properties which can limit bacterial attachment.

Adsorption of alpha-1-microglobulin from biological fluids onto polymer surfaces.

A recent study in our laboratory has identified the potential role of urine-derived alpha-1-microglobulin (alpha-1-m) in mediating Pseudomonas aeruginosa adhesion to polystyrene, while other workers have suggested a possible role of the protein in the immunological response. Due to the ubiquitous presence of alpha-1-m in body fluids, the adsorption of the protein from serum, cerebrospinal fluid, urine and used continuous ambulatory peritoneal dialysis fluid onto polystyrene was investigated. The treated surfaces were sequentially immersed in water and increasingly concentrated isopropanol-water solutions in order to selectively desorb bound proteins on the basis of their binding strength. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the wash supernatants showed different protein desorption profiles for each biological fluid, despite the qualitative similarity between the protein composition of the fluids, and highlighted the uptake of alpha-1-m from each fluid to the surface. In the case of urine, the analysis was extended to commercial polyurethane and silicone stents. The ease of desorption of urine-derived alpha-1-m could be correlated with surface hydrophobicity of the stent biomaterial.

Effect of the urine conditioning film on ureteral stent encrustation and characterization of its protein composition.

The goal of this study was to characterize the protein composition of the conditioning film deposited onto the surface of ureteral stents during in vivo implantation and to relate its presence to the precipitation of calcium crystals. The protein pattern of the conditioning film of implanted nonencrusted and encrusted urological stents was assessed by SDS-PAGE and Western blot of the desorbed species. The results obtained highlighted different electrophoresis profiles between nonencrusted and encrusted stents. Western blot showed the ubiquitous presence of albumin, while Tamm-Horsfall Protein and alpha1-microglobulin adsorption was limited to nonencrusted devices. By an in vitro dynamic model in which artificial urine was flowed through the lumen of control and retrieved nonencrusted stents, we demonstrated that the organic layer remarkably enhanced crystal precipitation and aggregation events on the surface.

Ocular biomaterials and implants.

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.

In vitro assessment of bacterial adhesion to Hydromer-coated cerebrospinal fluid shunts.

The adherence of five strains of Staphylococcus epidermidis and one strain of S. aureus to both untreated and Hydromer-coated silicone rubber cerebrospinal fluid shunts was studied in vitro using epifluorescent image analysis. All five strains of S. epidermidis showed similar levels of adherence to untreated shunts, whilst S. aureus adhered slightly better. The Hydromer coating, a hydrogel material which creates a hydrophilic layer on the shunt surface, was found to reduce bacterial adhesion levels by approximately 90% in the six strains of bacteria tested. Unfortunately, uniform coverage of the shunt surfaces (particularly internally) with Hydromer coating was not achieved during sample preparation. Bacterial adhesion levels in such areas were identical to untreated controls. This may pose problems in the in vivo use of Hydromer-coated shunts.

Uptake of drugs by catheters: the influence of the drug molecule on sorption by polyurethane catheters.

The sorption of drugs by indwelling intravenous catheters may have clinical consequences both by alteration of the dose received by the patient and by physically affecting the catheter materials themselves which may lead to changes in mechanical properties and biocompatibility. Studies of drug sorption to new catheter materials are therefore important. Pellethane, a polyurethane increasingly used in vascular access catheters, is as yet little studied in terms of its capacity for drug sorption. In this work a range of drugs known to be sorbed by PVC infusion sets were studied with respect to their sorption by Pellethane catheters. Standard lengths of catheter were incubated with solutions of drugs and samples of the solution were taken at intervals, assayed spectrophotometrically and compared with control solutions incubated without catheter. Losses from solution of up to 93% were found after 24 h. A series of highly sorbing and clinically relevant drugs was identified and their uptake was studied until equilibrium had been reached. A correlation was evident between the octanol/water partition coefficient and the fraction of drug taken up from solution at equilibrium, with the more hydrophobic drugs being taken up to a greater extent by the catheter.

A comparison of the use of an ATP-based bioluminescent assay and image analysis for the assessment of bacterial adhesion to standard HEMA and biomimetic soft contact lenses.

The aim of this study was to investigate in vitro adhesion of clinically relevant bacteria to standard HEMA and novel biomimetic soft contact lenses (SCL) using bioluminescent ATP assay and image analysis. Unworn SCL were incubated with Pseudomonas aeruginosa, Staphylococcus epidermidis or Serratia marcescens suspended in sterile phosphate buffered saline (PBS). The level of bacterial adhesion after 1, 2, 4, 6 and 18h, was assessed using both image analysis and a bioluminescent ATP assay. Species differences in the overall level of adhesion to the different types of lens were observed using both measurement techniques. Generally bacterial adhesion was shown to peak at 4-6 h, then decline to a much lower level by 18 h. After 4 h, adhesion of all species of bacteria to the biomimetic SCL (omafilcon A) was found to be significantly lower than to the standard HEMA SCL (polymacon) (p<0.05. Student's t-test, n = 4). Both these techniques demonstrated that novel biomimetic SCL materials exhibit significantly lower bacterial adhesion in vitro compared to standard HEMA SCL materials. SCL manufactured with these novel biomimetic materials may reduce the risk of infection.

Studies to show that with podophyllotoxin the early replicative stages of herpes simplex virus type 1 depend upon functional cytoplasmic microtubules.

The antiviral activity of podophyllotoxin against herpes simplex type 1 virus (HSV-1) grown in Vero cells was studied by a simple microtitration assay. Antiviral effects were induced at similar concentrations as direct cellular toxicity, as characterised by a time-dependent loss of cell monolayer. Podophyllotoxin-mediated toxicity arises from cytoplasmic microtubular, and hence cytoskeletal, decay. Some degree of selectivity was seen for inhibition of virus replication over direct cellular toxicity. Podophyllotoxin acted against an early viral process, as an antiviral effect was still seen if drug was removed 2 h after infection. Similar effects were seen with colchicine, a classical tubulin-binding compound, but not with bromovinyldeoxyuridine. Podophyllotoxin was capable of inducing a cytoprotective effect in Vero cells, as pre-treatment of cells abrogated virus growth for up to 90 min after removal of drug. This is coincident with the repolymerisation of cellular microtubules and re-formation of the cytoskeleton. We conclude that HSV-1 relies upon a functional cellular cytoskeleton for efficient completion of an early replicative event. Such a process may be the transport of viral material to the nucleus or inhibition of the formation of intranuclear viral 'replication factories', bodies containing cytoskeletal fragments constructed after viral infection.

Preservative activity in diluted corticosteroid creams.

The preservative efficacy of both 'Betnovate' and 'Synalar' creams diluted 1:1 with 'Unguentum Merck' was investigated. Each formulation was challenged with Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at an initial inoculum level of approximately 1 X 10(6) viable organisms per gram of cream. All formulations tested were found to be effectively preserved against the organisms used and no viable bacteria were detected 7 days after inoculation.