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.

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.

Evaluation of new in vitro efficacy test for antimicrobial surface activity reflecting UK hospital conditions.

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 24h 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.

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.

Evaluation of the bactericidal efficacy of three different alcohol hand rubs against clinical isolates of Staphylococcus aureus using an ex vivo carrier test.

We tested the efficacy of three alcohol hand rubs (AHRs) against Staphylococcus aureus using an ex vivo carrier test method and investigated the residual activity of AHRs and the effect of mechanical rubbing. A much longer contact time was required for the AHRs to achieve a bactericidal effect using the ex vivo test (between 10 and >20 min) compared with the in vitro test. Mechanical rubbing was found to increase the efficacy of the AHR compared to a rubbing control. Since the AHRs had no residual activity, the bactericidal effect achieved using the ex vivo test with contact times greater than the evaporation times (15 s) is unlikely to be achieved in practice. In view of such findings it is unlikely that AHRs are able to achieve a significant bactericidal effect (≥4 log(10) reduction) in practice, suggesting that contamination on the hands of healthcare workers (HCWs) may not be reduced enough to overcome the risk of cross-contamination and healthcare-associated infection. Since the AHRs had no residual effect they would be unable to prevent recolonisation of the hands of HCWs.

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.

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.

Evaluation of the bactericidal efficacy of three different alcohol hand rubs against 57 clinical isolates of S. aureus.

We tested the efficacy of three alcohol hand rubs (AHRs) used in two local Welsh intensive therapy units (ITUs) against Staphylococcus aureus. The test protocol was based on a carrier test and parameters (concentration, contact time) were chosen following observation of hand-sanitising practices in the ITUs. Following AHR exposure, surviving bacteria were enumerated using a standard plate count method plus a Bioscreen C Microbial Growth Analyser. The AHRs demonstrated variable efficacy against the clinical isolates: the mean log(10) reduction after 10 s exposure to Soft Care Med H5, Cutan and Guest Medical AHRs was 2.67, 0.696 and 1.96, respectively, and after 30 s exposure was 4.58, 1.74 and 3.60, respectively. Since the average time taken by healthcare workers (HCWs) to rub AHR onto their hands was 11 s and 15 s at the two hospitals, the efficacy of these AHRs may be significantly limited against the S. aureus isolates under the conditions observed in practice. In addition, differences observed in log(10) reduction in bacterial number post-exposure using the Bioscreen compared to the plate count method provided evidence that S. aureus may be able to recover following Guest Medical AHR treatment within 2 min exposure, whereas after 5 min exposure bacterial damage caused by the AHR was irreversible. Although the introduction of AHRs improved hand hygiene compliance among HCWs, our observations highlighted that contact time is an important factor to ensure the efficacy of these products.

Triclosan-tolerant bacteria: changes in susceptibility to antibiotics.

There is no clear consensus regarding the effect of biocide tolerance on antibiotic susceptibility. In this work, triclosan-tolerant strains of Escherichia coli, Staphylococcus aureus and Acinetobacter johnsonii were compared with sensitive strains in order to ascertain their susceptibility to a range of antibiotics. The minimum inhibitory concentrations of triclosan were measured using broth- and agar-dilution techniques. Antibiotic susceptibilities were determined using the British Society for Antimicrobial Chemotherapy guidelines. No triclosan-tolerant strains were resistant to antibiotics, and there was no overall tendency for triclosan-tolerant strains to have significantly smaller zones of inhibition compared with counterpart strains. Triclosan-tolerant strains of E. coli were significantly more susceptible to aminoglycoside antibiotics. The mechanism by which E. coli develops tolerance to triclosan appears to be linked to aminoglycoside susceptibility. It is proposed that changes in outer membrane, or the loss of plasmids, may be responsible for this relationship.

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.

Cell behaviour on phospholipids-coated surfaces.

Effective integration of orthopedic biomaterials requires the rapid formation of the inorganic mineral phase during the first hours of implantation and the subsequent adhesion and proliferation of the osteoblasts. It has recently been demonstrated that phosphatidylserine-rich phospholipid coatings can induce a fast mineralisation of titanium implant surfaces on incubation in simulated body fluids. The aim of this work was to investigate the biocompatibility of these coatings in terms of cytotoxicity and ability to support osteoblast adhesion and activity. Cytotoxicity and cell adhesion to uncoated titanium, calcified phospholipid-coated titanium and HA-coated titanium was assessed using fibroblasts and osteoblast-like cells. The synthesis of type I collagen by osteoblast-like cells cultured on the calcified-phospholipid coatings was also comparable to that observed for osteoblast-like cells cultured on the titanium and HA-Ti surfaces.The results suggest that the fast mineralization of the phospholipid matrix, obtained in vitro by its pre-treatment in a SBF, exposes the cells to an environment similar to that present in the bone during its natural formation that allow cells to adhere, proliferate and produce proteins fundamental for bone growth. The biocompatibility of these phospholipid-based coatings, in combination with their ability to initiate rapid mineralisation, provides a promising material that could in vivo create bone cell interactions and bone integration.

A simulated oral hygiene model to determine the efficacy of repeated exposure of amine oxide on the viability of Streptococcus mutans biofilms.

The use of biocide in oral care products is important for controlling microbial pathogens. However, the use of biofilm tests that investigate repeated exposure to biocide, to mimic in situ treatment, has rarely been reported in the literature. The present study describes the application of a biofilm-based efficacy protocol, for testing the effect of repeated exposure to antimicrobials on biofilm, in an attempt to mimic oral care regimens. The activity of different treatment regimens, including repeated exposure to amine oxide (AO; C(10)-C(16)-alkyldimethyl N-oxides; 1.1% v/v), was conducted against 16-h Streptococcus mutans biofilms grown on hydroxyapatite disks. Single exposure to AO alone produced a 3 log(10) reduction in microbial count, but when combined with mechanical removal, a 5 log(10) reduction in microbial count was observed. Treatments incorporating repeated exposure to AO reduced the microbial count below the level of detection, even when exposure to AO was interspersed with recovery periods. The presence of organic load produced an additional 2 log(10) reduction in the microbial count. This study showed that the application of a biofilm-based efficacy protocol to mimic oral care regimens allowed the reproducible testing of repeated antimicrobial exposures against bacterial biofilm. In addition, AO was confirmed to be an excellent biocide for eliminating S. mutans biofilms and could therefore be beneficial in oral care formulations.

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).

In vivo assessment of the osteointegrative potential of phosphatidylserine-based coatings.

The successful implantation of titanium-based implants for orthopaedic and dental applications is often hindered because of their mobility, which arises because of a lack of direct binding of the metal surface to the mineral phase of the surrounding bone. Ceramic coatings, although ensuring the integration of the implant within the tissue, are unstable and carry risks of delamination and of failure. Recently, a novel biomimetic approach has been developed where porous titanium implants are coated with calcium-binding phospholipids able to catalyse the nucleation of discrete apatite crystals after only 30 min incubation in simulated body fluids. The present work assesses the osteointegrative potential of this new class of coatings in an in vivo rabbit model and compares its performance with those of bare porous titanium and hydroxyapatite-coated titanium. The data obtained show that phosphatidylserine-based coatings, whilst resorbing, drive the growing bone into apposition with the metal surface. This is in contrast to the case of bare titanium.