Inactivation of a diverse set of shiga toxin-producing Escherichia coli in ground beef by high pressure processing.

Shiga toxin-producing Escherichia coli (STEC) are regularly implicated in foodborne illness outbreaks and recalls of ground beef. In this study we determined the High Pressure Processing (HPP) D10 value (the processing conditions needed to reduce the microbial population by 1 log) of 39 STEC isolates, including the "big six" serovars, O104 and O157:H7. STEC isolates included those isolated from animals and environmental sources in addition to those associated with illness in humans. Individual STEC were inoculated into 80% lean ground beef and treated with HPP (350 MPa, 4 °C, up to 40 min). The mean D10 was 9.74 min, with a range of 0.89-25.70 min. The D10 of the STEC involved in human illness was 9.25 vs. 10.40 min for those not involved in human illness (p > 0.05). The presence or absence of genes encoding virulence factors (e.g. Shiga toxin 1 or 2, intimin, or enterohemolysin) had no effect on the HPP D10 (p > 0.05). The high D10 of some STEC involved in human illness should be considered in selecting HPP processing parameters for ground beef. This study demonstrates the heterogeneity of STEC resistance to HPP. Risk assessors and the food industry can use this information to provide safer meat products to consumers.

Effect of high pressure processing on the survival of Shiga toxin-producing Escherichia coli (Big Six vs. O157:H7) in ground beef.

High pressure processing (HPP) is a safe and effective technology for improving food safety. Non-O157:H7 Shiga Toxin-producing Escherichia coli (STEC) have been increasingly implicated in foodborne illness outbreaks and recalls, and the USDA Food Safety Inspection Service (FSIS) has designated them as adulterants in meat (e.g. ground beef). In this study we compared the inactivation of multi-isolate cocktails of E. coli O157:H7 versus the non-O157:H7 STEC "Big Six" (i.e. O26, O45, O103, O111, O121, and O145) in ground beef (83% lean) using HPP at refrigeration temperature (4-7 °C). A >5-log CFU/g inactivation of both the Big Six and O157:H7 cocktails were observed at 450 MPa for 15 min. In general, the Big Six cocktail was found more sensitive to pressure stress (p < 0.05). In contrast, HPP treatment at 250 MPa (30 min) inactivated only 2.3 log of the Big Six versus 1.0 log of O157:H7. HPP treatment at 350 MPa (30 min) inactivated 4.7 log of the Big Six vs. 3.2 log of O157:H7. Multiple-cycle HPP cycles (250 or 350 MPa, three 5 min treatments) did not result in a 5 log reduction of the non-O157:H7 or O157:H7 STEC. Our results indicate that HPP inactivation parameters which are effective for O157:H7 STEC can be used for the non-O157:H7 Big Six isolates in ground beef.

Inactivation of Shiga toxin-producing Escherichia coli in lean ground beef by gamma irradiation.

In this study the radiation resistance of 40 Shiga Toxin-Producing Escherichia coli (STEC) isolates which contained various combinations of the shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eae), and hemolysin (ehx) genes were determined. The STEC were suspended in lean ground beef and irradiated at 4 °C. D10 values, the radiation dose needed to reduce 1 log (90%) of a microorganism, ranged from 0.16 to 0.48 kGy, with a mean of 0.31 kGy for the 40 isolates. Isolates associated with illness outbreaks had a mean D10 of 0.27 kGy, while non-outbreak isolates had a mean D10 of 0.36 kGy (p < 0.05). The presence or absence of stx1, stx2, or both stx1 and 2 had no affect on D10 (p > 0.05). The presence (0.30 kGy) or absence (0.35 kGy) of ehx had no affect on D10 (p > 0.05). However, the mean D10 of isolates lacking eae (0.37 kGy) were significantly higher than those containing eae (0.27 kGy) (p < 0.05). There was no difference in D10 for isolates lacking eae regardless of whether or not they were associated with a foodborne illness outbreak (p > 0.05). It may be possible to use some of the STEC isolates which lacked eae, ehx, or both (D10 > 0.30) as avirulent surrogates in food irradiation research. The data presented in this study provides risk assessors data for metagenomic analysis as well as food and radiation processors with valuable information to control of STEC in meat.

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii.

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD(+)-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

Minding the gap: identifying values to enable public and patient involvement at the pre-commencement stage of research projects.

BACKGROUND: The University College Dublin (UCD) Public and Patient Invovlement (PPI) ignite program is focused on embedding PPI in health and social care related research, education and training, professional practice and administration. During a PPI knowledge sharing event challenges were noted during the pre-commencement stage of research projects. This stage includes the time before a research projects/partnership starts or when funding is being applied for. As a response, we agreed there was a need to spend time developing a values-based approach to be used from the pre-commencement of PPI projects and partnerships. Values are deeply held ideals that people consider to be important. They are vital in shaping our attitudes and motivating our choices and behaviours. METHODS: Using independent facilitators, we invited a diverse group of participants to a full-day workshop in February. During the workshop, the concept of a values statement and values-based approaches was introduced. The group via a majority consensus, agreed on a core set of values and a shared understanding of them. After the workshop, a draft was shared with participants for further comment and final agreement. RESULTS: The workshop had 22 people representing experts by experience, PPI charity partners, funders, academics and national PPI Ignite partners. The group via consensus identified four values of respect, openness, reciprocity and flexibility for the pre-commencement stage. A frequently reported experience of PPI partners was that some felt that the pre-commencement activities appeared at times like a performance; an act that had to be completed in order to move to the next stage rather than a genuine interest in a mutually beneficial partnership. Being open and transparent with all invovled that the funding application may not be successful was stressed. Another important feature related to 'openness' was the 'spaces' and 'places' in which meetings between partners could occur in an accessible and equitable way. The issue of 'space' is particularly critical for the involvement of seldom heard groups. The benefits of the research are often clear for academics, but for PPI partners, these are often less certain. To achieve reciprocity, academic and PPI partners need to engage in a timely, repeated and transparent dialogue to achieve beneficial outcomes for all stakeholders. Being open to new inputs and differing modes of knowledge and ideas was also stressed. For some, this will require a change in attitudes and behaviours and should result in more collective decision making. Several areas were identified using the four values. CONCLUSIONS: This work via majority consensus identified four values of respect, openness, reciprocity, and flexibility for the pre-commencement stage. These values should be used to support inclusive, effective and collective PPI across all stages of involvement. We hope this work will stimulate further action in this area. In particular, we would welcome the evaluation of these values involving diverse PPI groups.

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii.

Xi class glutathione transferases (GSTs) are a recently identified group, within this large superfamily of enzymes, specifically endowed with glutathione-dependent reductase activity on glutathionyl-hydroquinone. Enzymes belonging to this group are widely distributed in bacteria, fungi, and plants but not in higher eukaryotes. Xi class GSTs are also frequently found in archaea and here we focus on the enzyme produced by the extreme haloalkaliphilic archaeon Natrialba magadii (NmGHR). We investigated its function and stability and determined its 3D structure in the apo form by X-ray crystallography. NmGHR displays the same fold of its mesophilic counterparts, is enriched in negatively charged residues, which are evenly distributed along the surface of the protein, and is characterized by a peculiar distribution of hydrophobic residues. A distinctive feature of haloalkaliphilic archaea is their preference for γ-glutamyl-cysteine over glutathione as a reducing thiol. Indeed we found that the N. magadii genome lacks a gene coding for glutathione synthase. Analysis of NmGHR structure suggests that the thiol binding site (G-site) of the enzyme is well suited for hosting γ-glutamyl-cysteine.

Haloquadratum walsbyi Yields a Versatile, NAD+/NADP+ Dual Affinity, Thermostable, Alcohol Dehydrogenase (HwADH).

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10% (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, and maximum activity was detected with benzyl alcohol and 2-phenyl-1-propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1-phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts.

Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

Effects of heat treatment on the antioxidative and anti-inflammatory properties of orange by-products.

This study investigated the changes in the functional components, antioxidative activities, antibacterial activities, anti-inflammatory activities of orange (Citrus sinensis (L.) Osbeck) by-products (OBP) on heat treatment at 50 and 100 °C (hereafter denoted 50D and 100D extracts, respectively). Optimal heating conditions were also investigated. The total phenolic content, flavonoid content and antioxidative activities of OBP extracts significantly increased on heat treatment. The lag time of Cu2+-induced oxidation of human LDL was increased by 2.61, 8.61 and 8.76-fold with the addition of 0.6, 0.8 and 1.0 mg ml-1 100D extracts, respectively. The 100D extracts may significantly inhibit the growth of E. coli O157, Salmonella typhimurium and Listeria monocytogenes. 1 µg mL-1 of 100D extract may suppress the TNF-α-induced ICAM-1 protein expression. The optimal heating time for OBP was 26 h at 100 °C, which resulted in the highest antioxidant activities.

Survival of Salmonella Typhimurium on soybean sprouts following treatments with gaseous chlorine dioxide and biocontrol Pseudomonas bacteria.

Control of Salmonella Typhimurium on sprouts is crucial for food and consumer safety. In this study, natural microflora on soybean seed was assessed and effects of gaseous chlorine dioxide (ClO2) and biocontrol Pseudomonas on the survival of S. Typhimurium on soybean sprouts were evaluated. Sprouts were dip-inoculated with S. Typhimurium prior to the application of the biocontrol (P. chlororaphis and P. fluorescens). After inoculation with S. Typhimurium, the sprouts were treated with ClO2 at 0.4 mg/L for 1 h (90% R.H., 13°C). Pseudomonas strains and Salmonella were recovered on Pseudomonas Agar F (PAF) and xylose lysine tergitol-4 (XLT-4) media, respectively. Pseudomonas strains reduced Salmonella by <1 log colony forming units (CFU)/g of sprouts, whereas S. Typhimurium on soybean sprouts was reduced from 2.55 to 5.35 logs CFU/g by ClO2. Gaseous ClO2 treatment reduced S. Typhimurium by 3.90 (0 h), 4.47 (24 h), and 3.61 log CFU/g (168 h). It was concluded that ClO2 and biocontrol treatment can enhance sprout safety.

Inactivation of Salmonella enterica and Listeria monocytogenes in cantaloupe puree by high hydrostatic pressure with/without added ascorbic acid.

The objective of this research was to evaluate and develop a method for inactivation of Salmonella enterica and Listeria monocytogenes in cantaloupe puree (CP) by high hydrostatic pressure (HHP). Cantaloupe being the most netted varieties of melons presents a greater risk of pathogen transmission. Freshly prepared CP with or without 0.1% ascorbic acid (AA) was inoculated with a bacterial cocktail composed of a three serotype mixture of S. enterica (S. Poona, S. Newport H1275 and S. Stanley H0558) and a mixture of three strains of L. monocytogenes (Scott A, 43256 and 51742) to a population of ca. 10(8)CFU/g. Double sealed and double bagged inoculated CP (ca. 5g) were pressure treated at 300, 400 and 500MPa at 8°C and 15°C for 5min. Data indicated increased inactivation of both Salmonella and Listeria spp. with higher pressure. Log reduction for CP at 300MPa, 8°C for 5min was 2.4±0.2 and 1.6±0.5logCFU/g for Salmonella and Listeria, respectively. Survivability of the pathogens was significantly compromised at 400MPa and 8°C, inactivating 4.5±0.3logCFU/g of Salmonella and 3.0±0.4logCFU/g of Listeria spp. Complete inactivation of the pathogens in the puree (log reduction >6.7logCFU/g), with or without AA, was achieved when the pressure was further increased to 500MPa, except that for Listeria containing no AA at 8°C. Listeria presented higher resistance to pressure treatment compared to Salmonella spp. Initial temperatures (8 and 15°C) had no significant influence on Salmonella log reductions. Log reduction of pathogens increased but not significantly with increase of temperature. AA did not show any significant antimicrobial activity. Viable counts were about 0.2-0.4logCFU/g less in presence of 0.1% AA. These data validate that HHP can be used as an effective method for decontamination of cantaloupe puree.

Heterologous overexpression, purification and characterisation of an alcohol dehydrogenase (ADH2) from Halobacterium sp. NRC-1.

Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.

Molecular characterization of cephalosporin-resistant Salmonella enterica serotype Newport isolates from animals in Pennsylvania.

Multidrug-resistant (MDR) strains of Salmonella enterica serotype Newport have been described for many years. However, the recognition of Newport strains with resistance to cephalosporin antibiotics is more recent. Plasmid-mediated CMY-2 AmpC beta-lactamases have been identified in Salmonella in the United States, and the bla(CMY-2) gene has been shown to be present in Salmonella serotype Newport. This organism is currently undergoing epidemic spread in both animals and humans in the United States, and this is to our knowledge the first description of the molecular epidemiology of this Salmonella strain in animals. Forty-two isolates were included in this study. All isolates were characterized by pulsed-field gel electrophoresis, plasmid analysis, and antibiogram. Four pulsed-field profiles with XbaI were observed. Plasmid analyses showed that although the majority of isolates harbored a single plasmid of 140 kb, this plasmid was not identical in all strains. All isolates showed the presence of the bla(CMY) gene by PCR. Integrons were detected in 16 of the 42 isolates; a fragment of approximately 1,000 bp, amplified with the intI-F and aadAI-R primers, confirmed the presence of the aadAI gene cassette within an integron in these 16 isolates. The potential for coselection of the bla(CMY) gene, if located on an MDR replicon, may not be dependent on any particular antibiotic but rather may be the result of more general antimicrobial use. If this replicon is mobile, it is to be expected that similar MDR strains of additional Salmonella serotypes will be recognized in due course.