Peter H. Cooke

Analyses of the Red-Dry-Rough Phenotype of an Escherichia coli O157:H7 Strain and Its Role in Biofilm Formation and Resistance to Antibacterial Agents

ABSTRACT In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.

Rheology and microstructure of low-fat Mozzarella cheese

The contributions of fat and moisture content to Mozzarella cheese texture were investigated to provide a basis for developing low-fat cheese with consumer acceptability. The characteristics of low-fat high-moisture (LFHM) experimental Mozzarella cheeses before and after 6 weeks of refrigerated storage were compared with those of high-fat low-moisture controls. High levels of either moisture in nonfat substance or fat in dry matter (FDM) were accompanied by decreases in hardness, complex viscosity, and elastic modulus and increases in meltability during the storage time. Starter culture bacteria were observed at the surface of the fat droplets, the latter having a tendency to coalesce during storage. Development of texture and meltability in LFHM Mozzarella appeared to be directly related to increased proteolysis of αs1-casein observed during storage. These results show the feasibility of making Mozzarella cheese containing <25% FDM with textural properties similar to those of a full-fat cheese if the product contains enough moisture and is stored under refrigeration for several weeks.

Factors influencing attachment of Escherichia coli O157:H7 to beef tissues and removal using selected sanitizing rinses

Attachment of E. coli O157:H7 and E. coli K12 to beef tenderloin filet, chuck, and adipose tissues was studied. Most attachment occurred within 1 min of incubation; the number of attached organisms depended on the concentration of bacteria in the liquid inoculum. Similar levels of E. coli bound to the three types of beef tissues tested. E. coli O157:H7 was heavily piliated; however, there was no significant difference between levels of bound E. coli O157:H7 and E. coli K12, indicating that these surface structures apparently are not involved in attachment. Scanning electron photomicrographs of meat tissue and of purified collagen suggested that bacteria attached primarily to collagen fibers. Rinsing solutions consisting of 10% trisodium phosphate (TSP), 2% acetic acid (HAc), phosphate-buffered saline (PBS) and combinations of each were tested for effectiveness in reducing the number of attached E. coli . The level of bacteria removed from tenderloin tissue following TSP, HAc, or PBS rinses did not differ considerably. When beef tissues were stored at 4°C for 18 h after the various rinse combinations, TSP rinse treatments reduced the levels of E. coli K12 and O157:H7 attached to adipose tissue up to 3.4 and 2.7 log units, respectively, compared to PBS rinse treatments. Therefore, TSP may be effective for reducing populations of E. coli O157:H7 on beef carcass tissue.

Environmental Influences on Bovine κ-Casein: Reduction and Conversion to Fibrillar (Amyloid) Structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of κ-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the κ-casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem.17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-κ). Surprisingly, at only 37°C, the RCM-κ-casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM-κ-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and κ turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in κ-casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM-κ-casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.

Global Structure of Microwave-Assisted Flash-Extracted Sugar Beet Pectin

The global structure of microwave-assisted flash-extracted pectins isolated from fresh sugar beet pulp has been studied. The objective was to minimize the disassembly and possibly the degradation of pectin molecules during extraction. These pectins have been characterized by high-performance size exclusion chromatography with light scattering, viscometric detection, and atomic force microscopy (AFM). Analysis of molecular parameters was performed on 15 and 8 microm size column packings. Samples analyzed with 15 microm packing gave weight-average molar masses that ranged from 532,000 to 1.2 million Da, radii of gyration from about 35 to 51 nm, polydispersities from 1.78 to 2.58, intrinsic viscosities from about 3.00 to 4.30 dL/g, and recoveries from 8.40 to 14.81% of dry weight. Chromatography revealed that a bimodal distribution of high molar mass spherical particles and lower molar mass coils was obtained. AFM images of pectin corroborated this conclusion and further revealed that these strands and spherical particles were integrated into networks. It is demonstrated that microwave-assisted extraction of sugar beet pulp under moderate pressure and at relatively low temperature could extract under acid conditions high molar mass, moderate-viscosity pectin in minutes rather than hours as required by conventional heating.

Thermal Stabilization of Collagen Fibers by Calcification

Mineralized collagenous tissue is known to be more stable than soft collagenous tissue both mechanically and thermally. We find that the denaturation temperature of collagen in bone scanned in differential scanning calorimetry at 5 degrees C/min is 155 degrees C, 90 degrees C higher than that in skin. Furthermore, when the bone is partially demineralized with citrate, a discrete intermediate denaturation temperature appears at 113 degrees C, indicating that the mineral is retained at preferential binding sites in the collagen until it is completely leached out. It is shown by electron microscopy that these sites are located in the overlap regions of the collagen fibrils. Collagen in bovine hide can be synthetically mineralized in vitro by impregnation with calcium acid phosphate solution, followed by raising the pH, causing the phosphate to precipitate. Some of the collagen in this synthetically calcified tissue has an elevated denaturation temperature, 104 degrees C. This temperature compares favorably with collagen that is tanned with chromium oxide-sulfate complexes. Calcium phosphate in synthetically mineralized hide, hydroxyapatite in bone, and chromium oxide-sulfate complexes in leather might share the same mechanism of thermal stabilization.

Escherichia coli O157:H7 Biofilm Formation on Romaine Lettuce and Spinach Leaf Surfaces Reduces Efficacy of Irradiation and Sodium Hypochlorite Washes

Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Biofilm-associated pathogens are relatively resistant to chemical treatments, but little is known about their response to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated with E. coli O157:H7 and stored at 4 degrees C for various times (0, 24, 48, 72 h) to allow biofilms to form. After each time, leaves were treated with either a 3-min wash with a sodium hypochlorite solution (0, 300, or 600 ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75, or 1 kGy). Viable bacteria were recovered and enumerated. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 1.3 log CFU/g for baby spinach and 1.8 log CFU/g for Romaine. Increasing time in storage prior to chemical treatment had no effect on spinach, and had an inconsistent effect on 600 ppm applied to Romaine. Allowing time for formation of biofilm-like aggregations reduced the efficacy of irradiation. D(10) values (the dose required for a 1 log reduction) significantly increased with increasing storage time, up to 48 h postinoculation. From 0 h of storage, D(10) increased from 0.19 kGy to a maximum of 0.40 to 0.43 kGy for Romaine and 0.52 to 0.54 kGy for spinach. SEM showed developing biofilms on both types of leaves during storage. Bacterial colonization of the stomata was extensive on spinach, but not on Romaine. These results indicate that the protection of bacteria on the leaf surface by biofilm formation and stomatal colonization can reduce the antimicrobial efficacy of irradiation on leafy green vegetables.

Inactivation of Escherichia coli O157:H7 and Salmonella by Gamma Irradiation of Alfalfa Seed Intended for Production of Food Sprouts†

Inonizing irradiation was determined to be a suitable method for the inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seed to be used in the production of food sprouts. The radiation D (dose resulting in a 90% reduction of viable CFU) values for the inactivation of Salmonella and E. coli O157:H7 on alfalfa seeds were higher than the D-values for their inactivation on meat or poultry. The average D-value for the inactivation of Salmonella on alfalfa seeds was 0.97 +/- 0.03 kGy; the D-values for cocktails of meat isolates and for vegetable-associated isolates were not significantly different. The D-values for nonoutbreak and outbreak isolates of E. coli O157:H7 on alfalfa seeds were 0.55 +/- 0.01 and 0.60 +/- 0.01 kGy, respectively. It was determined that the relatively high D-values were not due to the low moisture content or the low water activity of the seed. The D-values for Salmonella on alfalfa seeds from two different sources did not differ significantly, even though there were significant differences in seed size and water activity. The increased moisture content of the seed after artificial inoculation did not significantly alter the D-value for the inactivation of Salmonella. The results of this study demonstrate that 3.3- and 2-log inactivations can be achieved with a 2-kGy dose of ionizing radiation, which will permit satisfactory commercial yields of sprouts from alfalfa seed contaminated with E. coli O157:H7 and Salmonella, respectively.

Identification of extensin protein associated with sugar beet pectin.

Several studies have suggested that the emulsification properties associated with pectin obtained from sugar beet (Beta vulgaris) are due to the presence of a protein-pectin complex. Nevertheless, the identity of the protein has remained elusive. Pectin, extracted from sugar beet pulp by microwave-assisted extraction, and a commercial sample were both subjected to protease digestion with trypsin. The resulting peptides were separated from the pectin solution by ultrafiltration using a 3 kDa molecular weight cutoff (MWCO) membrane and analyzed using matrix-assisted laser desorption ionization with tandem time-of-flight mass spectrometry. The partial sequences derived from the mass spectrometry analyses of the resulting tryptic peptides are found to be highly consistent with extensin protein matched from the B. vulgaris Genetic Index database and also correspond to previously reported extensin peptides found in sugar beet cell suspension cultures. Further attempts were made to disassociate the protein from pectin using 1 M NaCl and a 100 kDa MWCO membrane; however, no peptides were observed following trypsin digestion of the permeate solution. This evidence suggests the existence of a complex between the pectin and extensin that is not due to ionic interactions. Trypsin digestion of commercial sugar beet pectin also produced the peptide profile observed with the microwave-assisted extracted pectin sample. Atomic force microscopy established that the number of rod-like elements decreased following protease treatment compared to the untreated sample.

Progressive dissociation of pectin.

The structural organization of alkaline soluble peach pectin was investigated over size ranges extending from micrometers to tenths of nanometers. Analysis was by electron microscopy and high-performance anion-exchange chromatography (HPAEC). Superimposed and individual circular microgels in the micrometer size range were isolated from mesocarp tissue of cell walls and visualized by rotary shadowing. Dilute NaCl and 50% aqueous glycerol disaggregated these microgels into rods, segmented rods, and kinked rods, which collectively comprised the internal gel network of the microgels. Image analysis of the shadowed specimens before and after disaggregation followed by curve fitting of the smoothed distributions revealed a multimodal distribution of lengths. HPAEC revealed that the multimodal aggregates were stable for the most part to further dissociation by increasing ionic strength.