Natural inactivation of Escherichia coli in anaerobic and reduced groundwater.

AIMS: Inactivation rates of Escherichia coli in groundwater have most often been determined in aerobic and oxidized systems. This study examined E. coli inactivation rates in anaerobic and extremely reduced groundwater systems that have been identified as recharge zones. METHODS AND RESULTS: Groundwater from six artesian wells was diverted to above-ground, flow-through mesocosms that contained laboratory grown E. coli in diffusion chambers. All groundwater was anaerobic and extremely reduced (ORP < -300 mV). Cells were plated onto mTEC agar during 21-day incubation periods. All data fit a bi-phasic inactivation model, with >95% of the E. coli population being inactivated <11·0 h (mean k = 0·488 ±0·188 h(-1) ). CONCLUSIONS: The groundwater geochemical conditions enhanced the inactivation of E. coli to rates approx. 21-fold greater than previously published inactivation rate in groundwater (mean k = 0·023 ± 0·030 h(-1) ). Also, mTEC agar inhibits E. coli growth following exposure to anaerobic and reduced groundwater. SIGNIFICANCE AND IMPACT OF THE STUDY: Aquifer recharge zones with geochemical characteristics observed in this study complement above-ground engineered processes (e.g. filtration, disinfection), while increasing the overall indicator micro-organism log-reduction rate of a facility.

Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer.

BACKGROUND: Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. METHODS: An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. RESULTS: At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. CONCLUSION: These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

Inhibiting Eph kinase activity may not be "Eph"ective for cancer treatment.

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by "leaky" or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

The occurrence of lysogenic bacteria and microbial aggregates in the lakes of the McMurdo Dry Valleys, Antarctica.

The McMurdo Dry Valleys of Antarctica form the coldest and driest ecosystem on Earth. Within this region there are a number of perennially ice-covered (3-6 m thick) lakes that support active microbial assemblages and have a paucity of metazoans. These lakes receive limited allochthonous input of carbon and nutrients, and primary productivity is limited to only 6 months per year owing to an absence of sunlight during the austral winters. In an effort to establish the role that bacteria and their associated viruses play in carbon and nutrient cycling in these lakes, indigenous bacteria, free bacteriophage, and lysogen abundances were determined. Total bacterial abundances (TDC) ranged from 3.80 x 10(4) to 2.58 x 10(7) cells mL(-1) and virus-like particle (VLP) abundances ranged from 2.26 x 10(5) to 5.56 x 10(7) VLP mL(-1). VLP abundances were significantly correlated (P < 0.05) with TDC, bacterial productivity (TdR), chlorophyll a (Chl a), and soluble reactive phosphorus (SRP). Lysogenic bacteria, determined by induction with mitomycin C, made up between 2.0% and 62.5% of the total population of bacteria when using significant decreases and increases in TDC and VLP abundances, respectively, and 89.5% when using increases in VLP abundances as the sole criterion for a successful induction event. The contribution of viruses released from induced lysogens contributed <0.015% to the total viral production rate. Carbohydrate and protein based organic aggregates were abundant within the water column of the lakes and were heavily colonized by bacteria and VLPs. Alkaline phosphatase activity was detected within the matrix of the aggregates, implying phosphorus deficiency and consortial nutrient exchanges among microorganisms.

Students' perspectives on the educational environment, Faculty of Medical Sciences, Trinidad

The Dundee Ready Education Environment Measure (DREEM) was administered to 70 final-year medical students and 36 first-year medical interns (pre-registration house officers). The overall total mean DREEM scores for the five subscales--namely, students' perceptions of the atmosphere, students' perceptions of learning, students' social self-perceptions, students' perceptions of teachers and students' academic self-perceptions--was 109.9 and the total mean scores for the subgroups--male students, male interns, female students and female interns--were 103.39, 111.82, 111.33 and 113.15, respectively. The lowest scores were assigned to students' social self-perceptions and students' perceptions of the atmosphere. All of the participants except the male interns recorded the highest scores for the subscale academic self-perceptions.

The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7.

A suite of fluorescent intracellular stains and probes was used, in conjunction with viable plate counts, to assess the effect of chlorine disinfection on membrane potential (rhodamine 123; Rh123 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3)), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride; CTC) and substrate responsiveness (direct viable counts; DVC) in the commensal pathogen Escherichia coli O157:H7. After a 5 min exposure to the disinfectant, physiological indices were affected in the following order: viable plate counts > substrate responsiveness > membrane potential > respiratory activity > membrane integrity. In situ assessment of physiological activity by examining multiple targets, as demonstrated in this study, permits a more comprehensive determination of the site and extent of injury in bacterial cells following sublethal disinfection with chlorine. This approach to assessing altered bacterial physiology has application in various fields where detection of stressed bacteria is of interest.

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms.

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7.

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms.

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Pre-employment screening of NHS employees with epilepsy.

A survey was carried out on whether district health authorities have adopted clear policies with regard to the pre-employment screening of people with epilepsy. The survey revealed that only a small number have done so and that a few others claimed to be following recommended guidelines. It is evident that there is a need for an organization, such as the Association of National Health Service Occupational Physicians, to develop a more authoritative and comprehensive set of guidelines in this area. The paper concludes by suggesting some of the key elements to be included in such a set of guidelines.

Restoration of wild-type pathogenicity to an attenuated DNA polymerase mutant of herpes simplex virus type 1.

The drug-resistant variant, RSC-26, which was derived from the herpes simplex virus type 1 wild-type strain SC16, expresses an altered DNA polymerase and has reduced pathogenicity in animal models. To determine whether the attenuation in pathogenicity was due solely to mutation in the polymerase gene, a fragment of the wild-type gene was cloned, transferred into the genome of RSC-26 and recombinants were isolated. Three recombinants examined had similar properties to wild-type virus with respect to their sensitivity to antiviral drugs, DNA polymerase activities and their pathogenicity for mice. These results strongly suggest that expression of the altered polymerase of RSC-26 results in attenuated pathogenicity.