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Biomedical subjects

C W Keevil

Publications and source records attributed to C W Keevil.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1999

Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments.

Chloroform-soluble material was extracted from two strains of L. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24 degreesC in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.

Colony Count, Microbial↗
PubMed1998

Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and -rRNA staining.

In water microcosm experiments, the survival times of Campylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10 degrees C (202 and 176 h, respectively) than at 22 and 37 degrees C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacter isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4 degrees C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30 degrees C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4 degrees C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a Campylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.

Animals↗
PubMed1997

A study of iron acquisition mechanisms of Legionella pneumophila grown in chemostat culture.

We recently demonstrated that the virulence of a clinical isolate of Legionella pneumophila is significantly attenuated when cultured in an iron-limited environment. In this study the influence of iron limitation on the expression of enzyme activities and iron-transport mechanisms was investigated. Expression of the important pathogenicity factor, the zinc metalloprotease, was reduced fivefold in response to iron limitation. Ferric citrate reductase activity was demonstrated in both iron-limited and replete cell fractions. Activity was located principally in the cytoplasm and periplasm, and was not enhanced by iron restriction. Optimum activity was observed with NADPH as reductant. Siderophores were not elaborated under these culture conditions. Iron-loaded transferrin enhanced the growth of steady-state, iron-limited cultures, demonstrating that transferrin represents a potentially important iron source for L. pneumophila in vivo. Although cell surface transferrin receptors were not detected, in vitro experiments demonstrated digestion of transferrin by the zinc metalloprotease activity of culture supernatants.

Bacterial Proteins↗
PubMed1997

A simple artificial urine for the growth of urinary pathogens.

A simple artificial urine medium (AUM) has been developed which provides conditions similar to that found in human urine. AUM solidified with agar enabled the recovery of a wide range of urease-positive and -negative urinary pathogens. Liquid AUM supported growth at concentrations of up to 10(8) cfu ml-1, as found in normal urine. Reproducible, steady-state growth also occurred over many generations in continuous culture. AUM was capable of forming crystals and encrustations resembling those found in natural urinary tract infections. The medium is a suitable replacement for normal urine for use in a wide range of experiments modelling the growth and attachment of urinary pathogens in the clinical environment.

Bacteriological Techniques↗
PubMed1996

Examination of biofilm formation and risk of infection associated with the use of urinary catheters with leg bags.

Urinary catheters and legs bags were simultaneously colonized by Escherichia coli and Proteus vulgaris using a model urinary drainage system. the system was continuously supplied with filter-sterilized artificial urine using a diurnal flow pattern. The extent of colonization was determined by assessment of both planktonic and biofilm formation over time. Contamination of the catheters resulted in rapid colonization of the whole system within a 24 h period. Contamination of the leg bags resulted in an ascending biofilm formation over a four-day period. Results indicated that infection risk could be minimized by changing the catheter and leg bags at least once a week. The design of the leg bags was not found to influence the rate or extent of biofilm formation.

Animals↗
PubMed1995

Reporter genes and fluorescent probes for studying the colonisation of biofilms in a drinking water supply line by enteric bacteria.

Biofilms containing diverse microflora were developed on bitumen-painted steel and glass tiles suspended in a chemostat model of a water distribution system. Escherichia coli, taken from a naturally occurring biofilm, was transformed with a plasmid containing the anaerobically induced nirB promoter fused to the lacZ reporter gene. The resulting transformant, PRB1, was introduced into the chemostat. After 7 and 13 days, an E. coli strain with an anaerobically induced Lac+ phenotype was present in the biofilm. Development of an episcopic differential interference contrast technique combined with UV fluorescence microscopy enabled the simultaneous visualization of E. coli in the biofilm using a fluorescent probe to detect expression of the gusA reporter gene and a lacZ fluorescent probe to monitor anaerobic expression of beta-galactosidase from pnirB.

Biofilms↗
PubMed1995

A paint incorporating silver to control mixed biofilms containing Legionella pneumophila.

A three-stage chemostat containing a mixed consortium of microorganisms, including Legionella pneumophila, was used to determine the suitability of a silver-containing paint to control biofouling in water systems. The paint was efficient in controlling total surface colonisation by heterotrophic microorganisms and growth of the pathogen over a 2-week period. Biodiversity was limited in the presence of the silver paint and this was thought to help control L. pneumophila numbers. Glass control tiles suspended alongside the silver painted tiles also had reduced colonisation for the 2-week period, suggesting that low levels of silver leached from the paint surface. This loss of silver was confirmed since the inhibition of biofouling and inclusion of the pathogen was not maintained after the 2-week period. Although this paint was unsuitable for controlling biofouling over extended time periods, the data suggest that a reformulated paint or electrochemical method of introducing silver ions may be successful.

Biofilms↗
PubMed1995

Control of Legionella pneumophila in a hospital water system by chlorine dioxide.

Immuno-compromised patients are particularly susceptible to Legionnaires' Disease. After three cases of the disease occurred in a hospital, a continuous dosing regime using chlorine dioxide was initiated to replace chlorination of the water system. This study identified a number of factors which may have resulted in conditions that would encourage the growth of the water-borne pathogen Legionella pneumophila. The residual chlorination was inadequate for microbial control at the taps furthest from the four storage tanks, of which two were found to be in excess for demand. The temperature of the water in the storage tanks was also found to be above 20 degrees C; a temperature that would encourage microbial growth. A back-up calorifier was present and was found to contain L. pneumophila, and linseed oil-based sealants that provide nutrients for microbial growth were also prevalent as jointing compounds in the water circuit. Although the shower heads were routinely disinfected, a requirement was identified to also disinfect the shower hoses. No L. pneumophila were recovered from the water system after the chlorine reduced dioxide disinfection trial. Biofilm was also dramatically reduced after disinfection; however, small microcolonies were identified and proved to be metabolically active when tested with a metabolic indicator. Using light and fluorescence microscopy, the pipe samples removed from the water system were rapidly analysed for biofouling, complementing existing microbiological methods.

Chlorine↗