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C W Keevil

Publications and source records attributed to C W Keevil.

At least 19 recordsLinked to original sources
PubMed2006

Are surgical stainless steel wires used for intracranial implantation of PrPsc a good model of iatrogenic transmission from contaminated surgical stainless steel instruments after cleaning?

Transmissible spongiform encephalopathies are a group of fatal, neurodegenerative diseases commonly known as prion diseases. Prion diseases can resist traditional inactivation strategies and may be iatrogenically transmitted by surgical instruments through the human population. These properties have led to the need for a suitable detection method of the prion infectious agent, and increased pressure regarding the development of anti-prion cleaning methodologies that would ensure the safety of surgical instruments. Although other techniques have been applied, the animal bioassay remains the 'gold standard' method for assessing infectivity. As the vast majority of surgical instruments are made of stainless steel, and in order to test this surface using the animal bioassay, the application of very thin surgical stainless steel wires has been widely adopted. These wires are easily inoculated and may be reimplanted into animals without the requirement for elution of the residual material. However, their comparability to the dimensions, shape and size of surgical instruments is questionable. This article shows how such contaminated wires (residual protein between 6.3 and 16.0 ng/mm(2)) can be cleaned more easily than flat metal surfaces (residual protein between 63.9 and 89.3 ng/mm(2)) under comparable conditions using recommended cleaning agents. These results indicate that the application of wires as a realistic means of assessing the removal or inactivation of the prion infectious agent from surgical instruments should be treated with caution.

Animals↗
PubMed2006

The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission.

Regulations recommend the routine application of biochemical tests, such as the Ninhydrin or Biuret tests, to confirm the efficacy of hospital sterile service department (SSD) washer-disinfector cycles in removing proteinaceous material, particularly with respect to prions. The effectiveness of these methods relies on both the effective sampling of the instruments and the sensitivity of the tests employed. Two commercially available contamination assessment tests were evaluated for their sensitivity to ME7 brain homogenate on surgical-grade stainless steel surfaces. Controls were visualized by the application of episcopic differential interference contrast/Epi-fluorecence microscopy (EDIC/EF) combined with the sensitive fluorescent reagent, SYPRO Ruby, which has been shown previously to rapidly visualize and assess low levels of contamination on medical devices. The Ninhydrin test displayed a minimum level of detection observed by 75% of volunteers (MLD(75)) of 9.25 microg [95% confidence interval (95% CI) 8.6-10.0 microg]. The Biuret test provided better sensitivity, with a MLD(75) of 6.7 microg (95% CI 5.4-8.2 microg). However, much lower concentrations of proteinaceous soiling (pg) were visualized using the EDIC/EF microscopy method. From these findings, it is clear that these approved colorimetric tests of cleaning are relatively insensitive. This investigation demonstrates how large amounts (up to 6.5 microg) of proteinaceous brain contamination could remain undetected and the instruments deemed clean using such methods. The application of more sensitive cleanliness evaluation methods should be applied to reduce the risk of iatrogenic transmission of prion disease in 'high-risk' instruments such as neurosurgical devices.

Biuret Reaction↗
PubMed2006

Comparative study of surgical instruments from sterile-service departments for presence of residual gram-negative endotoxin and proteinaceous deposits.

The ineffective cleaning of surgical instruments may be a vector for the transmission of hospital-acquired infections. The aim of this research was to investigate whether further decontamination procedures need to be instigated in sterile-service departments (SSDs) to reduce the risk of nosocomial illnesses, such as endotoxemia, sepsis, or iatrogenic Creutzfeldt-Jakob disease (to date, 1,147 cases of confirmed Creutzfeldt-Jakob disease deaths in the United Kingdom since 1990 have been reported). Instrument sets were obtained from nine anonymous United Kingdom National Health Service (NHS) primary care trust SSDs. The investigation implemented an advanced light microscopy technique, episcopic differential interference contrast microscopy with the sensitive fluorescent reagents SYPRO Ruby and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), to detect proteinaceous and microbial contamination levels. Gram-negative lipopolysaccharide (LPS) endotoxin was monitored using a dansylated polymyxin B fluorochrome agent. None of the 260 instruments examined displayed signs of microbial colonization or LPS endotoxin contamination. However, over 60 percent of the instruments showed a high degree of protein soiling (0.4 to 4.2 mug protein/mm(2)). Some instruments appeared soiled with crystalline deposits that may consist of a potentially hazardous material contributing to inflammation and/or surgical shock. It is clear that the overall standard for cleaning must be raised in order to fulfill the imminent introduction of new European standards and to reduce the risk of cross-patient contamination and iatrogenic transmission.

Decontamination↗
PubMed2006

Potential use of copper surfaces to reduce survival of epidemic meticillin-resistant Staphylococcus aureus in the healthcare environment.

Epidemic meticillin-resistant Staphylococcus aureus (EMRSA) emerged in the early 1980s with EMRSA-15 and -16 being the most prevalent strains within the UK. MRSA transmission between patients is largely via the hands of healthcare workers, and contamination of the hospital environment may occur. The objective of this study was to evaluate the effectiveness of copper and brass to reduce the viability of air-dried deposits of three MRSA strains [MRSA (NCTC 10442), EMRSA-1 (NCTC 11939) and EMRSA-16 (NCTC 13143)] compared with stainless steel. MRSA and EMRSA [10(7)colony-forming units (CFU)] were inoculated on to coupons (1 cm x 1 cm) of copper, brass or stainless steel and incubated at either 22 degrees C or 4 degrees C for various time periods. Viability was determined by resuspending removed CFUs and plating out on tryptone soy agar plates in addition to staining with the respiratory indicator fluorochrome 5-cyano-2,3-ditolyl tetrazolium. On pure copper surfaces, 10(7) MRSA, EMRSA-1 and EMRSA-16 were completely killed after 45, 60 and 90 min, respectively, at 22 degrees C. In contrast, viable organisms for all three strains were detected on stainless steel (grade 304) after 72 h at 22 degrees C. At 4 degrees C, complete kill was achieved on copper for all three strains within 6 h. The results demonstrate an antimicrobial effect of copper on MRSA, EMRSA-1 and -16 in contrast to stainless steel. Consequently, the contemporary application of stainless steel in hospital environments for work surfaces and door furniture is not recommended.

Colony Count, Microbial↗
PubMed2006

Drinking water biofilm assessment of total and culturable bacteria under different operating conditions.

Monitoring of biofilms subjected to different operating conditions was performed using a flow cell system. The system was fed by chlorine-free tap water, with and without added nutrients (0.5 mg l(-1) carbon, 0.1 mg l(-1) nitrogen and 0.01 mg l(-1) phosphorus), and biofilms were grown on polyvinyl chloride (PVC) and stainless steel (SS) coupons, both in laminar and turbulent flow. The parameters analysed were culturable cells, using R2A, and total bacteria, which was assessed using the 4,6-diamino-2-phenylindole (DAPI) staining method. The impact of the different operating conditions in the studied parameters was established using Multivariate Analysis of Variance (MANOVA). From the most relevant to the least relevant factor, the total and culturable bacteria in biofilms increased due to the addition of nutrients to water (F = 20.005; p < 0.001); the use of turbulent (Re = 11000) instead of laminar (Re = 2000) hydrodynamic flows (F = 9.173; p < 0.001); and the use of PVC instead of SS as the support material (F = 2.848; p = 0.060). Interactions between these conditions, namely between surface and flow (F = 8.235; p < 0.001) and also flow and nutrients (F = 5.498; p < 0.05) have also proved to significantly influence biofilm formation. This work highlights the need for a deeper understanding of how the large spectrum of conditions interact and affect biofilm formation potential and accumulation with the final purpose of predicting the total and culturable bacteria attached to real drinking water distribution pipes based on the system characteristics.

Biofilms↗
PubMed2006

Adhesion of water stressed Helicobacter pylori to abiotic surfaces.

AIM: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase. METHODS AND RESULTS: The number of total cells adhered for different water exposure times and modifications in the cell shape were evaluated using epifluorescence and scanning electron microscopy, and physiology assessed using Syto9 and propidium iodide (PI) cellular uptake. All abiotic surfaces were rapidly colonized by H. pylori, and colonization appeared to reach a steady state after 96 h with levels ranging from 2.3 x 10(6) to 3.6 x 10(6) total cells cm(-2). Cell morphology was largely dependent on the support material, with spiral bacteria, associated with the infectious form of H. pylori, subsisting in a higher percentage on nonpolymeric substrata. Also, sessile bacteria were generally able to retain the spiral shape for longer when compared with planktonic bacteria, which became coccoid more quickly. The formation of large aggregates, which may act as a protection mechanism against the negative impact of the stressful external environmental conditions, was mostly observed on the surface of copper coupons. However, Syto9 and PI staining indicates that most of H. pylori attached to copper or SS304 have a compromised cell membrane after only 48 h. Cultivability methods were only able to detect the bacteria up to the 2 h exposure-time and at very low levels (up to 500 CFU cm(-2)). CONCLUSIONS: The fact that the pathogen is able to adhere, retain the spiral morphology for longer and form large aggregates when attached to different plumbing materials appeared to point to pipe materials in general, and copper plumbing in particular, as a possible reservoir of virulent H. pylori in water distribution systems. However, the Syto9/PI staining results and cultivability methods indicate that the attached H. pylori cells quickly enter in a nonviable physiological state. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study of H. pylori behaviour in water-exposed abiotic surfaces. It suggests that co-aggregation with the autochthonous heterotrophic consortia present in water is necessary for a longer survival of the pathogen in biofilms associated to drinking water systems.

Bacterial Adhesion↗
PubMed2006

Use of copper cast alloys to control Escherichia coli O157 cross-contamination during food processing.

The most notable method of infection from Escherichia coli O157 (E. coli O157) is through contaminated food products, usually ground beef. The objective of this study was to evaluate seven cast copper alloys (61 to 95% Cu) for their ability to reduce the viability of E. coli O157, mixed with or without ground beef juice, and to compare these results to those for stainless steel. E. coli O157 (NCTC 12900) (2 x 10(7) CFU) mixed with extracted beef juice (25%) was inoculated onto coupons of each copper cast alloy or stainless steel and incubated at either 22 degrees C or 4 degrees C for up to 6 h. E. coli O157 viability was determined by plate counts in addition to staining in situ with the respiratory indicator fluorochrome 5-cyano-2,3-ditolyl tetrazolium. Without beef extract, three alloys completely killed the inoculum during the 6-h exposure at 22 degrees C. At 4 degrees C, only the high-copper alloys (>85%) significantly reduced the numbers of O157. With beef juice, only one alloy (95% Cu) completely killed the inoculum at 22 degrees C. For stainless steel, no significant reduction in cell numbers occurred. At 4 degrees C, only alloys C83300 (93% Cu) and C87300 (95% Cu) significantly reduced the numbers of E. coli O157, with 1.5- and 5-log kills, respectively. Reducing the inoculum to 10(3) CFU resulted in a complete kill for all seven cast copper alloys in 20 min or less at 22 degrees C. These results clearly demonstrate the antimicrobial properties of cast copper alloys with regard to E. coli O157, and consequently these alloys have the potential to aid in food safety.

Alloys↗
PubMed2006

Shear stress, temperature, and inoculation concentration influence the adhesion of water-stressed Helicobacter pylori to stainless steel 304 and polypropylene.

Although molecular techniques have identified Helicobacter pylori in drinking water-associated biofilms, there is a lack of studies reporting what factors affect the attachment of the bacterium to plumbing materials. Therefore, the adhesion of H. pylori suspended in distilled water to stainless steel 304 (SS304) coupons placed on tissue culture plates subjected to different environmental conditions was monitored. The extent of adhesion was evaluated for different water exposure times, using epifluorescence microscopy to count total cell numbers. High shear stresses-estimated through computational fluid dynamics-negatively influenced the adhesion of H. pylori to the substrata (P < 0.001), a result that was confirmed in similar experiments with polypropylene (P < 0.05). However, the temperature and inoculation concentration appeared to have no effect on adhesion (P > 0.05). After 2 hours, H. pylori cells appeared to be isolated on the surface of SS304 and were able to form small aggregates with longer exposure times. However, the formation of a three-dimensional structure was only very rarely observed. This study suggests that the detection of the pathogen in well water described by other authors can be related to the increased ability of H. pylori to integrate into biofilms under conditions of low shear stress. It will also allow a more rational selection of locations to perform molecular or plate culture analysis for the detection of H. pylori in drinking water-associated biofilms.

Bacterial Adhesion↗
PubMed2005

Rapid method for the sensitive detection of protein contamination on surgical instruments.

Hospital sterile service departments (SSDs) currently rely on simple visual confirmation of cleanliness as an assessment of the efficacy of cleaning surgical instruments. The inherent inability to monitor low levels of infectious or proteinaceous contamination on surgical instruments creates the possibility that highly dangerous and robust biological agents may remain infectious and undetected even after standard cleaning and sterilization procedures have been employed. This paper describes the development of a novel microscopy technique, episcopic differential interference contrast microscope, combined with the fluorescent reagent, SYPRO Ruby, to rapidly detect brain tissue protein to below 400 pg/mm(2) on an instrument surface. This technique has displayed a minimum level of detection observed by 50% of volunteers of 85 pg/mm(2) (95% confidence intervals 67-112 pg/mm(2)). Quantitative assessment of instruments supplied from various SSDs enabled the establishment of a 'contamination index' of both proteinaceous and non-proteinaceous deposits on the surface. This new methodology for the assessment of surface contamination is generally applicable and should facilitate future quantitative surveys of instrument contamination in hospitals and other healthcare environments.

Animals↗
PubMed2005

The survival of Escherichia coli O157 on a range of metal surfaces.

Escherichia coli O157:H7 is a serious pathogen causing haemorrhagic colitis. It has been responsible for several large-scale outbreaks in recent years. E. coli O157:H7 is able to survive in a range of environments, under various conditions. The risk of infection from contaminated surfaces is recognised, especially due to the low infectious dose required. In this study, a high concentration (10(7) cells) of E. coli O157 was placed onto different metals and survival time measured. Results showed E. coli O157 to survive for over 28 days at both refrigeration and room temperatures on stainless steel. Copper, in contrast, has strong antibacterial properties (no bacteria can be recovered after only 90 min exposure at 20 degrees C, increasing to 270 min at 4 degrees C) but its poor corrosion resistance and durability make it unsuitable for use as a surface material. Other copper-containing alloys, such as copper nickels and copper silvers, have improved durability and anticorrosion properties and greatly reduce bacterial survival times at these two temperatures (after 120 min at 20 degrees C and 360 min at 4 degrees C, no E. coli could be detected on a copper nickel with a 73% copper content). Use of a surface material with antibacterial properties could aid in preventing cross-contamination events in food processing and domestic environments, if standard hygiene measures fail.

Alloys↗
PubMed2004

Die-off of enteric bacterial pathogens during mesophilic anaerobic digestion.

Conventionally treated sewage sludge may contain high concentrations of potentially pathogenic microorganisms and additional treatment is required to minimise the risks to health if it is to be recycled to agricultural land. Mesophilic anaerobic digestion (MAD) is the most widely used process in the UK for stabilising sludge prior to agricultural recycling, but little is known about the fate of a number of enteric pathogens as the sludge passes through the treatment processes. The aim of this study was to determine the efficiency of MAD in removing the bacterial enteric pathogens, Salmonella senftenberg, Listeria monocytogenes and Campylobacter jejuni which were added as a spike to the digester feedstock, together with the die-off of indigenous Escherichia coli already present in the sludge. The primary sludge digestion stage of MAD was found to achieve a log removal of 1.66 for E. coli, 2.23 for L. monocytogenes and 2.23 for S. senftenberg. However, the extent of die-off was a function of the numbers of pathogens in the feed and as these increased the log removal also increased. The numbers of C. jejuni were not affected by primary sludge digestion. Additional die-off was provided by secondary sludge digestion with log removals of 1.70 for E. coli, 2.10 for S. senftenberg and 0.36 for C. jejuni.

Agriculture↗
PubMed2004

Nutrient shock and incubation atmosphere influence recovery of culturable Helicobacter pylori from water.

Three different media-Columbia agar, Wilkins-Chalgren agar, and Helicobacter pylori special peptone agar-were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering H. pylori from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed H. pylori organisms recovered. An atmosphere of 5% CO(2), 5% O(2), and 3% H(2) is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of H. pylori, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water.

Bacteriological Techniques↗
PubMed2004

The physico-chemistry of biofilm-mediated pitting corrosion of copper pipe supplying potable water.

Copper is a generally robust material that has beneficial properties to reduce biofilm formation and pathogen colonisation of pipes supplying potable water. However, a rare pitting corrosion can occur in soft, poorly buffered waters that can lead to pipe failure. This has been shown to be mediated by a copper-tolerant biofilm whose physical and chemical heterogeneity can establish microenvironments for corrosion potentials, causing micro pits that eventually coalesce into large perforations through the pipe wall. Control of the biofilm, for example through reduced cold water or elevated hot water temperatures, can suppress this corrosion phenomenon.

Biofilms↗
PubMed2003

Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy.

Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.

Animals↗
PubMed2003

Establishment of a continuous model system to study Helicobacter pylori survival in potable water biofilms.

Close association of the pathogen Helicobacter pylori in drinking water biofilms has been suggested. Using a two-stage water model, the survival and development of the pathogen in potable water biofilms was monitored. Filter sterilized tap water was used as the growth medium and the inoculum consisted of a naturally occurring consortium of microorganisms. Biofilms were generated on removable stainless steel coupons that were placed in the second vessel. Novel technology peptide nucleic acid (PNA) molecular probes were used to detect and locate the pathogen in the biofilms. The PNA-labelled oligonucleotide probes were highly specific, and complementary to the helix 6 region of H. pylori 16S rRNA. The pathogen was tracked in the biofilms using epifluorescence microscopy and episcopic differential interference contrast microscopy. Results show that H. pylori can successfully incorporate within biofilms and its presence was detected for up to five days after inoculation. PNA probes provided an easy and quick way of performing fluorescence in situ hybridisation assays in heterogeneous biofilms.

Biofilms↗
PubMed1999

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157.

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.

Aerobiosis↗