Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.     

An investigation of the thermal inactivation of Staphylococcus aureus and the potential for increased thermotolerance as a result of chilled storage

AIMS: The aims of this study were; (i) to provide thermal inactivation data for Staphylococcus aureus; (ii) to examine the kinetics, including decimal reduction times (D-value) and rate constants (k), that describe the thermal inactivation of Staph. aureus and to compare two different methods of calculating D-values and (iii) to determine whether or not chilled storage would toughen these microorganisms resulting in increased thermotolerance. METHODS AND RESULTS: Isolates of Staph. aureus recovered from domestic refrigerators were grown in shaken culture for 8 h at 37 degrees C, recovered and washed by centrifugation and combined to form a cocktail of five strains. Samples from this cocktail were (a) heat treated at 50, 55 and 60 degrees C or (b) held under simulated domestic refrigeration conditions for 72 h and then heat treated as above. The numbers of Staph. aureus in heat treated and chill held, heat treated samples were enumerated by direct selective plating onto Baird Parker Agar (BPA) and recovery plating on Tryptone Soya Agar (TSA) subsequently overlaid with BPA. D-values were obtained using two different methods both of which may be used when the thermal inactivation follows first order kinetics. In the first method D-values are obtained by plotting the Log(10) of the surviving cells against time and using the equation D = -1/slope. The second method uses the rate constant (k) which is obtained from the slope of a plot of ln N/N(0)vs time and D is obtained using the equation D = 2.303 k(-1). D(50), D(55) and D(60) values ranged from 94.3 to 127.9 min, 13 to 21.7 min and 4.8 to 6.5 min. Prechilling did not enhance thermal resistance. The method of calculation did not affect the D-values obtained because the thermal inactivation of Staph. aureus in this study followed first order kinetics with r(2) values of 0.91-0.99. CONCLUSIONS: The thermal inactivation of Staph. aureus in tryptone soya broth (TSB) follows first order kinetics and in general chilling of these bacteria does not increase the resistance to thermal destruction during subsequent thermal processes such as cooking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides much needed data on the thermal resistance of Staph. aureus and validates chilling as a food storage activity which does not cause toughening of the microorganisms to subsequent cooking. However, the data generated strongly suggests that Staph. aureus is more thermotolerant than Listeria monocytogenes and should be used as the target microorganism in designing mild thermal treatments for food, in which case the current recommendations for pasteurization (70 degrees C for 2 min, minimum) should be revised.     

Excision vs sponge swabbing - a comparison of methods for the microbiological sampling of beef, pork and lamb carcasses

AIMS: The aim of this research was to compare excision sampling with polyurethane and cellulose acetate sponge swabbing for the recovery of total viable counts and Enterobacteriaceae on meat carcasses. METHODS AND RESULTS: Two sample types were used to compare the methods: (i) individual samples, taken from four sites on each carcass and (ii) composite samples, created by pooling the samples from four sites from an additional set of carcasses. When the polyurethane sponge and excision method were compared for individual sites, there were no significant differences in bacterial recovery on beef and pork carcasses and on two of four sites on lamb carcasses. However, when samples from each site were pooled, the excision method was more efficient than either swabbing method across the three animal species. CONCLUSIONS: Sampling using the polyurethane sponge represents an equivalent alternative method to excision for the bacteriological sampling of carcass surfaces which is nondestructive and less labour intensive. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the scientific basis for using sponge swabbing instead of excision in compliance with 2001/471/EC.     

Spatial epidemiology and natural population structure of Campylobacter jejuni colonizing a farmland ecosystem

Recent progress in determining the population structure of Campylobacter jejuni, and discerning associations between genotypes and specific niches, has emphasized the shortfall in our understanding of the ecology and epidemiology of this bacterium. We examined the natural structure of the C. jejuni community associated with cattle farmland in the UK by structured spatiotemporal sampling of habitats, including livestock and wild animal faeces, environmental water and soil, over a 10-week period within a 100 km2 area. A total of 172 isolates were characterized using multilocus sequence typing into 65 sequence types (STs). Isolates from cattle faeces were significantly over-represented in the ST-61 complex, whereas isolates from wildlife faeces and water were more likely to belong to the ST-45 complex and a number of unusual STs, many of which were first encountered during this study. Sampling within a narrow spatiotemporal window permitted the application of novel statistical methods exploring the relationship between the genetic relatedness and spatial separation of isolates. This approach showed that isolates from the same sampling squares and squares separated by <1.0 km were genetically more similar than isolates separated by greater distances. Our study demonstrates the potential of multilocus sequence typing combined with spatial modelling in exploring natural transmission pathways for C. jejuni.     

Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus

It is well documented that long term potentiation (LTP) is impaired in the hippocampus of the aged animal. Among the changes that contribute to this impairment is an increase in hippocampal concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and increased IL-1beta-induced signaling. In this study we investigated the possibility that these changes were a consequence of decreased concentration of the anti-inflammatory cytokine, IL-4, and decreased IL-4-stimulated signaling. We report that functional IL-4 receptors are expressed on granule cells of the dentate gyrus and that receptor activation results in phosphorylation of JAK1 and STAT6. Hippocampal IL-4 concentration was decreased with age, and this was accompanied by a decrease in phosphorylation of JAK1 and STAT6. The evidence indicates that IL-4 modulates expression of IL-1beta mRNA and protein and that it attenuates IL-1beta-induced impairment of LTP and phosphorylation of JNK and c-Jun. We argued that, if a decrease in hippocampal IL-4 concentration significantly contributed to the age-related impairment in LTP, then restoration of IL-4 should restore LTP. To test this, we treated rats with VP015 (phospholipid microparticles-incorporating phosphatidylserine), which increases IL-4 concentration in hippocampus. The data indicate that the VP015-induced increase in IL-4 concentration in hippocampus of aged rats and lipopolysaccharide (LPS)-treated rats was accompanied by a reversal of the age-related and LPS-induced impairment in LTP in perforant path granule cell synapses. We propose that interplay between pro-inflammatory and anti-inflammatory responses impact significantly on synaptic function in the hippocampus of the aged rat.     

Subsite specificities of granzyme M: a study of inhibitors and newly synthesized thiobenzyl ester substrates

Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.     

Within‐year variation in the vegetative spread of five temperate grasses

Questions: Do species planted outdoors in native soil differ in the timing of their period of vegetative spread during the growing season? Location: Cleish Hills, Fife, Scotland, UK. Methods: Patches (20 cm × 20 cm) of Agrostis capillaris, Festuca rubra, Holcus lanatus, Lolium perenne and Poa trivialis were arranged in plots so that every species patch had all the remaining species as neighbours. The plots were cut every 14 days at 3 cm and photographed monthly from above. The photographs of species boundaries were traced and digitised. Measurements were made on the digitised images of the distances each species spread and of the area occupied by each species. Results: Fitting some simple models to the data indicated firstly that species varied in their vegetative competitive ability, with A. capillaris and P. trivialis being the most and least competitive respectively, and secondly that the ability of a species to colonize can differ from its ability to resist colonization, the most discrepant species being H. lanatus. Our analysis also indicated statistically significant variation in the data over and above the simple models, providing evidence of additional, genuine, complexity in the seasonal patterns of spread. Conclusion: This study provides evidence to support the hypothesis that clonal grass species do not spread vegetatively at the same times within a growing season. Since sward height is known to affect species spread, there is some opportunity for the manipulation of species composition simply through temporal control of sward height.     

Role of intracellular stores in the regulation of rhythmical [Ca2+]i changes in interstitial cells of Cajal from rabbit portal vein

Interstitial cells of Cajal (ICCs) freshly isolated from rabbit portal vein and loaded with the Ca(2+)-sensitive indicator fluo-3 revealed rhythmical [Ca(2+)](i) changes occurring at 0.02-0.1 Hz. Each increase in [Ca(2+)](i) originated from a discrete central region of the ICC and propagated as a [Ca(2+)](i) wave towards the cell periphery, but usually became attenuated before reaching the ends of the cell. In about 40% of ICCs each rhythmical change in [Ca(2+)](i) consisted of an initial [Ca(2+)](i) increase (phase 1) followed by a faster rise in [Ca(2+)](i) (phase 2) and then a decrease in [Ca(2+)](i) (phase 3); the frequency correlated with the rate of rise of [Ca(2+)](i) during phase 1, but not with the peak amplitude. Rhythmical [Ca(2+)](i) changes persisted in nicardipine, but were abolished in Ca(2+)-free solution as well as by SK&F96365, cyclopiazonic acid, thapsigargin, 2-APB, xestospongin C or ryanodine. Intracellular Ca(2+) stores visualised with the low-affinity Ca(2+) indicator fluo-3FF were found to be enriched with ryanodine receptors (RyRs) detected with BODIPY TR-X ryanodine. Rhythmical [Ca(2+)](i) changes originated from a perinuclear S/ER element showing the highest RyR density. Immunostaining with anti-TRPC3,6,7 antibodies revealed the expression of these channel proteins in the ICC plasmalemma. This suggests that these rhythmical [Ca(2+)](i) changes, a key element of ICC pacemaking activity, result from S/ER Ca(2+) release which is mediated via RyRs and IP(3) receptors and is modulated by the activity of S/ER-Ca(2+)-ATPase and TRP channels but not by L-type Ca(2+) channels.