Fruit Flies as Potential Vectors of Foodborne Illness.

Fruit flies are a familiar sight in many food service facilities. Although they have been long considered as "nuisance pests," some of their typical daily activities suggest they may pose a potential public health threat. The aim of this study was to provide evidence of the ability of small flies to transfer bacteria from a contaminated source, food, or waste to surfaces or ready-to-eat food. Laboratory experiments were conducted by using purpose-built fly enclosures to assess the bacterial transfer capability of fruit flies. Drosophila repleta were capable of transferring Escherichia coli O157:H7, Salmonella Saint Paul, and Listeria innocua from an inoculated food source to the surface of laboratory enclosures. In addition, using an inoculated doughnut and noncontaminated lettuce and doughnut surfaces, fly-mediated cross-contamination of ready-to-eat food was demonstrated. Fruit flies were shown to be capable of accumulating approximately 2.9 × 103 log CFU of E. coli per fly within 2 h of exposure to a contaminated food source. These levels of bacteria did not decrease over an observation period of 48 h. Scanning electron micrographs were taken of bacteria associated with fly food and contact body parts and hairs during a selection of these experiments. These data, coupled with the feeding and breeding behavior of fruit flies in unsanitary areas of the kitchen and their propensity to land and rest on food preparation surfaces and equipment, indicate a possible role for fruit flies in the spread of foodborne pathogens.

Gene expression profiling of Escherichia coli in response to interactions with the lettuce rhizosphere.

AIMS: The objective of this study was to examine transcriptional changes in Escherichia coli when the bacterium was growing in the lettuce rhizoshpere. METHODS AND RESULTS: A combination of microarray analyses, colonization assays and confocal microscopy was used to gain a more complete understanding of bacterial genes involved in the colonization and growth of E. coli K12 in the lettuce root rhizosphere using a novel hydroponic assay system. After 3 days of interaction with lettuce roots, E. coli genes involved in protein synthesis, stress responses and attachment were up-regulated. Mutants in curli production (crl, csgA) and flagella synthesis (fliN) had a reduced capacity to attach to roots as determined by bacterial counts and by confocal laser scanning microscopy. CONCLUSIONS: This study indicates that E. coli K12 has the capability to colonize lettuce roots by using attachment genes and can readily adapt to the rhizosphere of lettuce plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study show curli production and biofilm modulation genes are important for rhizosphere colonization and may provide useful targets to disrupt this process. Further studies using pathogenic strains will provide additional information about lettuce-E. coli interactions.

A microRNA gene expression signature predicts response to erlotinib in epithelial cancer cell lines and targets EMT.

BACKGROUND: Treatment with epidermal growth factor receptor (EGFR) inhibitors can result in clinical response in non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) for some unselected patients. EGFR and KRAS mutation status, amplification of EGFR, or gene expression predictors of response can forecast sensitivity to EGFR inhibition. METHODS: Using an NSCLC cell line model system, we identified and characterised microRNA (miRNA) gene expression that predicts response to EGFR inhibition. RESULTS: Expression of 13 miRNA genes predicts response to EGFR inhibition in cancer cell lines and tumours, and discriminates primary from metastatic tumours. Signature genes target proteins that are enriched for epithelial-to-mesenchymal transition (EMT) genes. Epithelial-to-mesenchymal transition predicts EGFR inhibitor resistance and metastatic behaviour. The EMT transcription factor, ZEB1, shows altered expression in erlotinib-sensitive NSCLC and PDAC, where many signature miRNA genes are upregulated. Ectopic expression of mir-200c alters expression of EMT proteins, sensitivity to erlotinib, and migration in lung cells. Treatment with TGFß1 changes expression of signature miRNA and EMT proteins and modulates migration in lung cells. CONCLUSION: From these data, we hypothesise that the tumour microenvironment elicits TGFß1 and stimulates a miRNA gene expression program that induces resistance to anti-EGFR therapy and drives lung tumour cells to EMT, invasion, and metastasis.

Fate of Escherichia coli O157:H7 in ground beef following high-pressure processing and freezing.

AIMS: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. METHODS AND RESULTS: Fresh ground beef was inoculated with a five-strain cocktail of E. coli O157:H7 vacuum-packaged, pressure-treated at 400 MPa for 10 min at -5 or 20 degrees C and stored at -20 or 4 degrees C for 5-30 days. A 3-log CFU g(-1) reduction of E. coli O157:H7 in the initial inoculum of 1 x 10(6) CFU g(-1) was observed immediately after pressure treatment at 20 degrees C. During frozen storage, levels of E. coli O157:H7 declined to <1 x 10(2) CFU g(-1) after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55-65 degrees C. CONCLUSIONS: High-pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g(-1) and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. SIGNIFICANCE AND IMPACT OF THE STUDY: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk.

AIMS: The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin. METHODS AND RESULTS: Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40 degrees C with or without 500 IU ml(-1) of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2.5 logs by cycled HP, while the addition of nisin (500 IU ml(-1)) prior to HP treatment resulted in log reductions of 5.7 and 5.9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40 degrees C in the presence of 500 IU ml(-1) nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed. CONCLUSIONS: Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure.

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.

Characterization of the interactions among vaccinia virus transcription factors G2R, A18R, and H5R.

Prior genetic analysis suggests that there may exist an interaction between the products of the vaccinia virus genes A18R, a putative negative transcription elongation factor, and G2R, a putative positive transcription elongation factor. In addition, affinity purification of polyhistidine-tagged G2R protein overexpressed in vaccinia virus-infected cells, reported here, results in copurification of the vaccinia H5R protein, previously characterized as a late viral transcription factor. We have therefore used several methods to screen further for interactions among the G2R, A18R, and H5R proteins. Methods include copurification or co-immunoprecipitation of proteins overexpressed during vaccinia virus infection, activation of the gal 4 promoter by gal 4 fusions in the yeast two-hybrid system, and co-immunoprecipitation of proteins synthesized in vitro in a rabbit reticulocyte lysate. The results reveal interactions which include all possible pairwise combinations of the three proteins G2R, A18R, and H5R; however, not all possible permutations of the interactions are observed and the interactions are not observed in all environments tested. The results suggest that the vaccinia virus proteins G2R, A18R, and H5R interact as part of a higher order transcription complex.

Phenotypic characterization of mutants in vaccinia virus gene G2R, a putative transcription elongation factor.

The phenotypic defects of two mutants of vaccinia virus, the lesions of which map to gene G2R, were characterized in vivo, and the results suggest a role for the G2R protein in viral transcription elongation. Both a temperature-sensitive mutant, Cts56, and an isatin-beta-thiosemicarbazone-dependent deletion mutant, G2A, in gene G2R have a characteristic and unique defect in late viral gene expression. The G2R mutants synthesize early viral RNA, early viral proteins, and viral DNA normally under nonpermissive conditions. In G2R mutants, late viral protein synthesis begins at the normal time, low-molecular-weight viral proteins are synthesized in normal quantities, but synthesis of high-molecular-weight viral proteins is reduced in amount. Intermediate and late promoter utilization is normal in G2R mutants, but intermediate and late RNAs are reduced in size. The reduction in length of the intermediate and late mRNAs represents a truncation of mRNA 3' ends. Thus, intermediate and late RNAs are too short to encode large proteins but long enough to encode small proteins, therefore accounting for the protein synthesis phenotype. These results suggest that the G2R protein acts to regulate the elongation potential of the viral RNA polymerase late during a vaccinia virus infection.

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.

Cytokines and serum cause alpha 2 beta 1 integrin-mediated contraction of collagen gels by cultured retinal pigment epithelial cells.

PURPOSE: Integrins, heterodimeric cell surface glycoproteins, are involved in cell-substratum and cell-cell interactions. The role of these molecules in cytokine-mediated contraction of extracellular matrix by retinal pigment epithelial cells has been investigated in a model system that may mimic epiretinal membrane contraction during retinal detachment in proliferative vitreoretinopathy. METHODS: Retinal pigment epithelial cells were cultured on three-dimensional collagen gels that they cause to contract. The involvement of new protein synthesis and C-kinase-mediated signal transduction were studied using specific inhibitors. Cell surface integrins synthesized by pigment epithelial cells were identified using immunofluorescence, and the same antibodies were used in gel contraction assays to identify the integrin species responsible for force transduction. RESULTS: Contraction of collagen type I gels was small without cytokines but was greatly enhanced in the presence of serum or IL1 plus TGF beta. It was dependent on new protein synthesis. Contraction induced by the combination of cytokines was dependent on active protein kinases, whereas that induced by serum was not because only the former was inhibited by staurosporine, a C-kinase inhibitor. Pigment epithelial cells were found to produce beta 1, alpha 2, and alpha 5 integrins, but only alpha 2 and beta 1 appeared to participate in the contraction process because only antibodies against these integrins inhibited contraction. CONCLUSIONS: Retinal pigment epithelial cells contract an extracellular matrix in vitro in a manner similar to epiretinal membrane contraction in vivo. This contraction is mediated via cell surface glycoproteins of the integrin family that bind directly to extracellular matrix molecules.

Cytokines cause cultured retinal pigment epithelial cells to secrete metalloproteinases and to contract collagen gels.

PURPOSE: Because retinal pigment epithelial cells in epiretinal membranes remodel and contract their surrounding extracellular matrix, investigations were performed to determine if these cells can produce matrix metalloproteinases and contract collagen gels in vitro in the presence of serum or cytokines. METHODS: Cells were grown on collagen gels and their production of metalloproteinases was measured using zymography. RESULTS: Cells grown in a three-dimensional collagen gel culture system produce several latent metalloproteinases that are secreted into the gel and the surrounding medium. These include molecules of 49, 56, 66, and 100 kD. In addition, an enzyme that is probably the active form of the 66 kD enzyme is present. When interleukin 1 beta is added to the cultures, latent 49 kD and 100 kD gelatinase production is greatly stimulated and an active form of both enzymes is also observed in the medium. In contrast, transforming growth factor beta has no stimulatory effect. The cells contract the collagen gel but this is small without cytokines; however, contraction is greatly enhanced in the presence of serum or interleukin 1 beta plus transforming growth factor beta. Contraction is unlikely to be the result of metalloproteinase action on the underlying extracellular matrix because complete inhibition of these enzymes has little effect. CONCLUSIONS: These results show that cytokines can cause cultured retinal pigment epithelial cells to produce metalloproteinases that can, when activated, degrade most collagens and other structural molecules in extracellular matrix. In addition, they can stimulate the contraction of extracellular matrix constituents but there is not a simple causal relationship between matrix remodeling and contraction.