Sensitive microscopic quantification of surface-bound prion infectivity for the assessment of surgical instrument decontamination procedures.

BACKGROUND: Pathogenic prions (PrPSc) are amyloid-rich hydrophobic proteins which bind avidly to surgical surfaces and represent some of the most difficult targets during the reprocessing of reusable surgical instruments. In-vitro methods to amplify and detect the presence of otherwise undetectable prion contamination are available, but they do not measure associated infectivity. Most of these methods rely on the use of proteinase K, however this can lead to the loss of a substantial portion of PrPSc, potentially producing false negatives. AIM: To develop a sensitive in-situ method without proteinase treatment for the dynamic quantification of amyloid accumulation in N2a #58 cells following 22L-prion infection from infected tissues and spiked stainless-steel surfaces. METHODS: We spiked cultures of N2a #58 cells with the 22L prion strain in solution or dried on stainless-steel wires and directly measured the accumulation of prion amyloid aggregates over several passages using highly sensitive fluorescence microscopy. FINDINGS: We demonstrated a 10-log dynamic range using our method to test residual prion infectivity, that was validated to show variable decontamination efficacy against prions from commercially available cleaning chemistries. CONCLUSIONS: The new cell-based infectivity method presented here avoids partial or possibly total proteinase K digestion of PrPSc in samples for greater sensitivity, in addition to low cost, no ethical concerns, and adaptability to detect different prion strains. This method can be used to test cleaning chemistries' efficacy with greater sensitivity than measuring total residual proteins, which may not correlate with residual prion infectivity.

A cold water, ultrasonically activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel.

BACKGROUND: Sterile service department decontamination procedures for surgical instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, such as prion proteins. A recently designed novel system, which uses a low pressure ultrasonically activated, cold water stream, has previously demonstrated efficient hard surface cleaning of several biological contaminants. AIM: To test the efficacy of an ultrasonically activated stream for the removal of tissue proteins, including prion-associated amyloid, from surgical stainless steel surfaces. METHODS: Test surfaces were contaminated with 22L, ME7 or 263K prion-infected brain homogenates. The surfaces were treated with the ultrasonically activated water stream for contact times of 5 and 10 s. Residual proteinaceous and amyloid contamination were quantified using sensitive microscopic analysis, and immunoblotting was used to characterize the eluted prion residues before and after treatment with the ultrasonically activated stream. FINDINGS: Efficient removal of the different prion strains from the surgical stainless steel surfaces was observed, and reduced levels of protease-susceptible and -resistant prion protein was detected in recovered supernatant. CONCLUSION: This study demonstrated that an ultrasonically activated stream has the potential to be a cost-effective solution to improve current decontamination practices and has the potential to reduce hospital-acquired infections.

Modelling vaporised hydrogen peroxide efficacy against mono-species biofilms.

This pilot study investigates a novel approach towards efficacy testing of antimicrobial cleaning agents; focusing primarily on hydrogen peroxide vapour (HPV). Contaminated surfaces are recognised modes of pathogen transmission within healthcare environments and increase the risk of pathogen acquisition in newly admitted patients. Studies have shown these pathogens can survive on surfaces for extended periods of time in spite of cleaning. This resilience is characteristic of biofilm formation and recent publications have identified their presence in hospitals. In this study, biofilm models comprised of multidrug-resistant organisms (MDROs) were generated using a drip flow reactor and exposed to HPV decontamination. The MDROs included Acinetobacter baumannii, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Upon exposure, samples were periodically removed and enumerated to generate kill curves for each species. Consequently revealing any inherent resistances; such as catalase-producing organisms which expressed reduced susceptibility. Epifluorescence microscopy revealed an abundance of viable and non-viable microcolonies before and after decontamination, respectively. Greater than 6-Log10 reduction was achieved within a 100 minutes exposure time. This pilot study puts forward a potential methodology for testing antimicrobial agents against biofilms and supports the efficacy of HPV.

Efficacy of humidity retention bags for the reduced adsorption and improved cleaning of tissue proteins including prion-associated amyloid to surgical stainless steel surfaces.

Increasing drying time adversely affects attachment of tissue proteins and prion-associated amyloid to surgical stainless steel, and reduces the efficacy of commercial cleaning chemistries. This study tested the efficacy of commercial humidity retention bags to reduce biofouling on surgical stainless steel and to improve subsequent cleaning. Surgical stainless steel surfaces were contaminated with ME7-infected brain homogenates and left to dry for 15 to 1,440 min either in air, in dry polythene bags or within humidity retention bags. Residual contamination pre/post cleaning was analysed using Thioflavin T/SYPRO Ruby dual staining and microscope analysis. An increase in biofouling was observed with increased drying time in air or in sealed dry bags. Humidity retention bags kept both protein and prion-associated amyloid minimal across the drying times both pre- and post-cleaning. Therefore, humidity bags demonstrate a cheap, easy to implement solution to improve surgical instrument reprocessing and to potentially reduce associated hospital acquired infections.

Improved recovery of Listeria monocytogenes from stainless steel and polytetrafluoroethylene surfaces using air/water ablation.

AIMS: To develop a gentle ablation technique to recover Listeria monocytogenes biofilms from stainless steel (SS) and polytetrafluoroethylene (PTFE) surfaces by using compressed air and water injection. METHODS AND RESULTS: Biofilms were grown for 4, 24 and 48 h or 7 days and a compressed air and water flow at 2, 3 and 4 bars was applied for cell removal. Collected cells were quantified for total/dead by staining with SYTO 9/PI double staining and cultivable populations were determined by plating onto brain heart infusion (BHI) agar, while coupon surfaces also were stained with DAPI to quantify in situ the remaining cells. The recovery efficiency was compared to that of conventional swabbing. Results showed that the air/water ablation is able to collect up to 98·6% of cells from SS surfaces while swabbing only recovered 11·2% of biofilm. Moreover, air/water ablation recovered 99·9% of cells from PTFE surfaces. CONCLUSIONS: The high recovery rate achieved by this technique, along with the fact that cells were able to retain membrane integrity and cultivability, indicate that this device is suitable for the gentle recovery of viable L. monocytogenes biofilm cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents a highly efficient technique to remove, collect and quantify L. monocytogenes from surfaces commonly used in the food industry, which can thus serve as an important aid in verifying cleaning and sanitation as well as in reducing the likelihood of cross-contamination events.

Hydrodynamic shear stress to remove Listeria monocytogenes biofilms from stainless steel and polytetrafluoroethylene surfaces.

AIMS: To calculate the shear stress needed to remove sessile Listeria monocytogenes cells from stainless steel (SS) and polytetrafluoroethylene (PTFE) surfaces. METHODS AND RESULTS: Listeria monocytogenes biofilms were formed on SS and PTFE surfaces. Shear stress was calculated using a radial flow chamber device and cells quantified by staining with 4',6-diamidino-2-phenylindole. Results showed that shear stress between 24 and 144 N m(-2) removed up to 98% of cells from SS surfaces. PTFE presents a very hydrophobic surface, and a significant lower removal (P < 0.05) of only 63% was achieved; moreover, on PTFE discs, detachment of L. monocytogenes biofilms was more efficient at a lower shear stress (between 8.6 and 34 N m(-2) ). CONCLUSIONS: Water flow is more effective in removing L. monocytogenes biofilms from SS surfaces than from PTFE materials. SIGNIFICANCE AND IMPACT OF THE STUDY: This work clearly demonstrates that water flow does not have the same efficiency in removing cells from different material surfaces and shows the need to optimize cleaning and sampling procedures by considering the conditions in which cells attach to surfaces and the physicochemistry of the surfaces.

Current limitations about the cleaning of luminal endoscopes.

BACKGROUND: The presence and potential build-up of patient material such as proteins in endoscope lumens can have significant implications, including toxic reactions, device damage, inadequate disinfection/sterilization, increased risk of biofilm development and potential transmission of pathogens. AIM: To evaluate potential protein deposition and removal in the channels of flexible luminal endoscopes during a simple contamination/cleaning cycle. METHODS: The level of contamination present on disposable endoscopy forceps which come into contact with the lumen of biopsy channels was evaluated. Following observations in endoscopy units, factors influencing protein adsorption inside luminal endoscope channels and the action of current initial cleaning techniques were evaluated using a proteinaceous test soil and very sensitive fluorescence epimicroscopy. FINDINGS: Disposable endoscope accessories appear to be likely to contribute to the contamination of lumens, and were useful indicators of the amount of proteinaceous soil transiting through the channels of luminal endoscopes. Enzymatic cleaning according to the manufacturer's recommendations and brushing of the channels were ineffective at removing all proteinaceous residues from new endoscope channels after a single contamination. Rinsing immediately after contamination only led to a slight improvement in decontamination outcome. CONCLUSION: Limited action of current decontamination procedures and the lack of applicable quality control methods to assess the cleanliness of channels between patients contribute to increasing the risk of cross-infection of potentially harmful micro-organisms and molecules during endoscopy procedures.

Down-regulation of DNA mismatch repair enhances initiation and growth of neuroblastoma and brain tumour multicellular spheroids.

Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.

Adsorption of prion and tissue proteins to surgical stainless steel surfaces and the efficacy of decontamination following dry and wet storage conditions.

Iatrogenic transmission of the infectious prion protein (PrP(Sc)) is a potential threat due to its resistance to many chemical and enzymatic decontamination protocols and its strong adhesive properties to stainless steel. The conditions in which surgical instruments are handled during and after surgery may affect the level of tissue protein, prion attachment and the efficacy of subsequent decontamination regimes. This study investigated the adhesion of tissue protein and prion-associated amyloid to surgical stainless steel with respect to time and various storage conditions, and the subsequent outcome on the efficacy of enzymatic cleaning chemistries. Surfaces were contaminated with ME7-infected brain homogenate and left to dry between 0 and 120 min at room temperature or 24 h, in dry or moist conditions. Residual contamination before and after cleaning was visualised using sensitive fluorescent staining and episcopic differential interference contrast/epifluorescence microscopy. Longer drying times increased both protein and prion amyloid adsorption and affected the efficacy of the cleaning chemistries tested. A moist environment post-contamination significantly reduced the attachment of both protein and prion amyloid to the surgical stainless steel surface. Maintaining moist conditions could potentially improve the subsequent decontamination of reusable surgical instruments, also reducing process time and cost.

Application of a fluorescent dual stain to assess decontamination of tissue protein and prion amyloid from surgical stainless steel during simulated washer-disinfector cycles.

Current World Health Organization guidelines pertaining to the reprocessing of surgical instruments in the face of potential iatrogenic transmission of Creutzfeldt-Jakob disease (iCJD) are incompatible for the vast majority of devices. This has led to the advent of a range of new decontamination measures. Even without the implementation of these new procedures, the incidence of proven iCJD through surgery remains low. In this study, existing decontamination processes in sterile service departments have been evaluated using simulated washer-disinfector cycles on surgical grade stainless steel wires inoculated with ME7 scrapie homogenate. The consequence of varying the soil drying times and choice of cycle pre-treatment on prion removal were evaluated. Assessment of residual contamination at each cycle phase was carried out with the application of a sensitive fluorescent staining procedure to identify both total protein and prion-associated amyloid. The study confirmed that immediate reprocessing following contamination was beneficial during the pre-treatment phase with either an enzymatic or pre-soak wetting agent. Final total protein levels at the end of the cycles, were not significantly different from those where the soil was allowed to dry. In addition, cycles involving a pre-treatment with either an enzymatic cleaner or pre-soak, whether the soil was allowed to dry or not, showed complete removal of detectable prion amyloid. The results suggest that current decontamination procedures, combined with immediate processing of surgical instruments, have the potential to be highly effective alone at reducing the risk of surgical transmission of CJD.

Validation of SYTO 9/propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila.

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires' disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l(-1)), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD BacLight). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l(-1) of free chlorine and in 10 min when the concentration is increased to 1.2 mg l(-1). However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.

Survival of Clostridium difficile on copper and steel: futuristic options for hospital hygiene.

Clostridium difficile is rapidly becoming a major cause of hospital-acquired infections worldwide, due in part to transmission of the faecal pathogen between contaminated hands and contact surfaces. Accordingly, this study evaluated survival of C. difficile vegetative cells and spores on the contact surface commonly found in healthcare settings, stainless steel, compared to five copper alloys (65-100% copper content). C. difficile requires prolonged incubation to grow and therefore the total number and number of viable cells was estimated using a fluorescence dual-staining technique. For viability assessment the redox dye 5-cyano-2,3-ditolyl tetrazolium (CTC) was used to measure metabolic activity. Results demonstrated that copper alloys with a copper content >70% provide a significant reduction in survival of C. difficile vegetative cells and spores on copper alloys compared with stainless steel. Complete death of spores was observed after 24-48 h on copper alloys whereas no significant death rate was observed on stainless steel even after 168 h. The use of CTC gave comparable results to culture and offers a more rapid viability analysis (8 h) than culture. The results suggest that using copper alloys in hospitals and other healthcare facilities could offer the potential to reduce spread of C. difficile from contaminated surfaces.

Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsy specimens.

In this work, a fluorescence in situ hybridization (FISH) method for the rapid detection of Helicobacter pylori using a novel peptide nucleic acid (PNA) probe is reported. Laboratory testing with several different bacterial species, including other Helicobacter spp., has shown that this probe is highly specific for H. pylori strains. In addition, the PNA FISH method has been successfully adapted for detection of the pathogen in paraffin-embedded gastric biopsy specimens.

Inactivation of influenza A virus on copper versus stainless steel surfaces.

Influenza A virus particles (2 x 10(6)) were inoculated onto copper or stainless steel and incubated at 22 degrees C at 50 to 60% relative humidity. Infectivity of survivors was determined by utilizing a defined monolayer with fluorescent microscopy analysis. After incubation for 24 h on stainless steel, 500,000 virus particles were still infectious. After incubation for 6 h on copper, only 500 particles were active.

Effect of drying time, ambient temperature and pre-soaks on prion-infected tissue contamination levels on surgical stainless steel: concerns over prolonged transportation of instruments from theatre to central sterile service departments.

Iatrogenic transmission of prions through use of surgical instruments has been shown both experimentally and clinically. In addition, recent discoveries of prion protein accumulation in peripheral tissues such as appendix and muscle, and evidence suggesting human-to-human blood-borne transmission, have led to a concern that any residual soiling containing this agent may remain infectious even after sterile service processing. Removal of all proteinaceous material from surgical devices is extremely important for effective sterilization. This removal can be severely hampered if the contaminant is allowed to dry onto the instrument surface for any length of time. The current move to centralize sterile service centres and the inevitable lengthening of transport time between theatres and re-processing makes it necessary to minimize the amount of residual soiling adhering to an instrument before sterilization. This investigation simulates the period between the application of surgical instruments in theatre and their initial pre-wash by a washer/disinfector. The aim was to investigate the kinetics of drying at different temperatures, and the application of different commercially available pre-soak solutions in situ. The findings show that all pre-soaks significantly reduce (by up to 96%) the prion-infected tissue contamination, and that controlling the temperature whilst in transit between theatres and cleaning facilities may allow an increase in time before high protein adsorption levels occur.

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.

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.

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.

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.

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.