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Biomedical subjects

W M Waites

Publications and source records attributed to W M Waites.

At least 19 recordsLinked to original sources
PubMed2003

The effect of acid shock on sporulating Bacillus subtilis cells.

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.

Arabidopsis Proteins↗
PubMed2003

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sprouts.

AIMS: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. METHODS AND RESULTS: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. CONCLUSIONS: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.

Aerobiosis↗
PubMed2002

Carton sterilization by u.v.-C excimer laser light: recovery of Bacillus subtilis spores on vegetable extracts and food simulation matrices.

AIMS: To determine the recovery of Bacillus subtilis spores loaded onto preformed cartons and irradiated with u.v.-excimer laser (248 nm) light. METHODS AND RESULTS: Bacillus subtilis spores irradiated with u.v.-excimer laser light retained phase brightness, but were blocked at various stages of germination. In the presence of germinant, the majority of spores began to lose phase brightness but only after an extended lag period (ca 90 min). After 6 h ca 9% of the spores had elongated but failed to form new cells, approx. 12% had undergone partial phase darkening (grey spores), 15% remained phase bright whilst the remainder had turned fully phase dark but failed to elongate. No enhanced recovery of u.v.-treated spores (with intact or permeabilized coats) occurred in media containing hen egg white lysozyme or vegetable extracts (celery, carrot, swede or turnip). However, recovery did occur when irradiated spores were incubated for 26 d, semiaerobically, within cartons containing nutrient broth or milk. CONCLUSIONS: The germination ability of B. subtilis spores is altered following u.v.-excimer laser treatment. Recovery of treated spores was found in liquid systems but not on agar plates supplemented with vegetable extracts or lysozyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential recovery of u.v.-excimer laser-treated spores in a range of carton-packed food systems requires further investigation.

Animals↗
PubMed2002

Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage.

AIMS: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk. METHODS AND RESULTS: Thirty-seven isolates of Ps. fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively. The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol x l(-1) calcium chloride at 20 degrees C. The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol x l(-1) EDTA. They were heat stable and retained activity even after treatment at 121 degrees C for 20 min. One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration. Attempts to purify the lipases proved unsuccessful. CONCLUSIONS: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments. However, the smaller proteases have not been described previously. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples.

Animals↗
PubMed2001

Attachment of bacteria to beef from steam-pasteurized carcasses.

The extent to which a bacterial cocktail containing equal numbers of Pseudomonas fragi NCTC 10689, Listeria monocytogenes BL5/2, Salmonella Typhimurium LT2, and Escherichia coli JM 109 attached to loin surface cuts (7 by 5 cm) derived from steam-pasteurized beef carcasses has been evaluated. The extent of attachment was categorized as loosely attached (removed by rinsing), firmly attached (released by stomaching), and irreversibly bound. No significant difference (P > 0.10) in the attachment of bacteria to steam-pasteurized carcasses was found compared with control loin samples that had received no treatment. No significant difference (P > 0.05) was also found in the attachment strength between the different bacterial species tested. Most bacteria inoculated onto the loin cuts were reversibly bound, since they had been removed by rinsing and stomaching. The irreversible attachment of bacteria to loin cuts was found to vary significantly (P < 0.01) among the different carcass sets used but was independent of whether the carcass had undergone steam pasteurization treatment. Use of a bioluminescent strain of E. coli showed that cells bound preferentially to cut edges and convoluted areas on the loin surface and could not be removed by rinsing. The possible mechanisms of bacterial attachment and the suitability of steam pasteurization to remove contamination incurred during slaughter are discussed.

Animals↗
PubMed2000

Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project.

Vegetables are frequent ingredients of cooked chilled foods and are frequently contaminated with spore-forming bacteria (SFB). Therefore, risk assessment studies have been carried out, including the following: hazard identification and characterisation--from an extensive literature review and expertise of the participants, B. cereus and C. botulinum were identified as the main hazards; exposure assessment--consisting of determination of the prevalence of hazardous SFB in cooked chilled foods containing vegetables and in unprocessed vegetables, and identification of SFB representative of the bacterial community in cooked chilled foods containing vegetables, determination of heat-resistance parameters and factors affecting heat resistance of SFB, determination of the growth kinetics of SFB in vegetable substrate and of the influence of controlling factors, validation of previous work in complex food systems and by challenge testing and information about process and storage conditions of cooked chilled foods containing vegetables. The paper illustrates some original results obtained in the course of the project. The results and information collected from scientific literature or from the expertise of the participants are integrated into the microbial risk assessment, using both a Bayesian belief network approach and a process risk model approach, previously applied to other foodborne hazards.

Bacillus cereus↗
PubMed2000

The growth of Listeria monocytogenes in cheese packed under a modified atmosphere.

The effect of modified atmosphere Packaging (MAP) on the growth of Listeria monocytogenes in mould ripened cheeses was studied at refrigeration temperatures (2-8.3 degrees C) over a storage period of 6 weeks. Control experiments in cling film with no atmospheric modification produced a lag time before growth of up to 1 week and rapid subsequent growth. MAP with a CO2 concentration of less than 20% allowed growth to occur but when O2 was incorporated; the lag time was reduced from 3 to 2 weeks and subsequent growth was also faster, producing an increase in cell numbers of 1.4 log cycles over the incubation period. N2-MAP in the absence of O2 increased the lag time to 3 weeks and slowed growth, while the inclusion of CO2 extended the lag to 3 weeks and slowed subsequent growth even more. In MAP with 80:10:10 (v/v/v) N2:CO2:O2, there was a lag period of 2-3 weeks before growth of L. monocytogenes occurred, while the total viable aerobic count (TVAC) decreased by 2-3 log cycles and the total Lactobacillus count showed little change. It was concluded that MAP was not suitable for preventing the growth of L. monocytogenes in such cheeses.

Carbon Dioxide↗
PubMed2000

Inactivation of Bacillus subtilis spores on packaging surfaces by u. v. excimer laser irradiation.

Ultraviolet (u.v.) laser irradiation has been used to inactivate Bacillus subtilis spores deposited on to planar aluminium- and polyethylene-coated packaging surfaces. Kill kinetics were found to be diphasic, with an initial rapid inactivation phase followed by tailing. Although no definitive evidence was obtained, it is thought that spores located within packaging crevices/pores were primarily responsible for the observed tailing. Surviving spores were also found on the unexposed underside of cards and, to a lesser extent, within clumps. The log count reduction in B. subtilis was dependent on spore loading and total u.v. dose. In comparison, packaging surface composition, fluence (2-18 Jm-2) and frequency (40-150 Hz) had only a negligible effect. By irradiating boards carrying 106 spores, with a dose of 11.5 J cm-2, a log count reduction >5 was obtained. The mode of spore inactivation was primarily through DNA disruption. This was confirmed by the high sensitivity of spores lacking protective, small, acid-soluble proteins, in addition to the high frequency of auxotrophic and asporogenous mutations found amongst survivors.

Bacillus subtilis↗
PubMed2000

Effect of flagella on initial attachment of Listeria monocytogenes to stainless steel.

At 22 degrees C a flagellin mutant of Listeria monocytogenes was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37 degrees C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.

Bacterial Adhesion↗
PubMed2000

Inactivation of Bacillus subtilis spores on aluminum and polyethylene preformed cartons by UV-excimer laser irradiation.

The efficacy of UV KrF-excimer laser light (at 248 nm) to inactivate Bacillus subtilis spores loaded onto preformed cartons was found to be dependent on the interior carton coating and scheme by which the irradiation was applied. When the carton was held static during UV laser treatment, the majority of the dose was delivered to the base of the carton and to a lesser extent to the upper part of the pack. In this arrangement no irradiation of the interior sides of the carton was observed. A more even distribution of dose was achieved, however, by moving the carton within the laser beam during irradiation treatment. The distribution of UV was also found to be dependent on the type of carton interior coating. With aluminum cartons the dose measured was found to be significantly greater (P < 0.01) and more evenly distributed across the interior compared to when polyethylene packs were tested. Under optimized conditions no spore survivors were detected on aluminum cartons preloaded with 9.5 x l0 B. subtilis spores by applying a UV laser output dose of 160 J. In comparison, the same conditions only achieved a significantly lower (P < 0.01) reduction in spore numbers (log count reduction 4.2) when polyethylene cartons were used. This difference in lethality and UV distribution of laser light was associated with the higher internal reflection of photons with aluminum cartons. The suitability of UV-excimer lasers for sterilizing preformed cartons over traditional germicidal lamp-based methods is discussed.

Aluminum↗
PubMed1999

Effects of combined shear and thermal forces on destruction of Microbacterium lacticum.

A twin-screw extruder and a rotational rheometer were used to generate shear forces in concentrated gelatin inoculated with a heat-resistant isolate of a vegetative bacterial species, Microbacterium lacticum. Shear forces in the extruder were mainly controlled by varying the water feed rate. The water content of the extrudates changed between 19 and 45% (wet weight basis). Higher shear forces generated at low water contents and the calculated die wall shear stress correlated strongly with bacterial destruction. No surviving microorganisms could be detected at the highest wall shear stress of 409 kPa, giving log reduction of 5.3 (minimum detection level, 2 x 10(4) CFU/sample). The mean residence time of the microorganism in the extruder was 49 to 58 s, and the maximum temperature measured in the end of the die was 73 degrees C. The D(75 degrees C) of the microorganism in gelatin at 65% water content was 20 min. It is concluded that the physical forces generated in the reverse screw element and the extruder die rather than heat played a major part in cell destruction. In a rotational rheometer, after shearing of a mix of microorganisms with gelatin at 65% (wt/wt) moisture content for 4 min at a shear stress of 2.8 kPa and a temperature of 75 degrees C, the number of surviving microorganisms in the sheared sample was 5.2 x 10(6) CFU/g of sample compared with 1.4 x 10(8) CFU/g of sample in the nonsheared control. The relative effectiveness of physical forces in the killing of bacteria and destruction of starch granules is discussed.

Actinomycetales↗
PubMed1996

Survival of Staphylococcus aureus and enterotoxin A in glassy and rubbery states of gelatin.

About 1% of Staphylococcus aureus cells survived the production of gelatin sheets containing nutrient broth. Those cells which survived showed no evidence of injury. Growth occurred in rubbery state gelatin with a(w) values of 0.98 and 0.93 ; viability decreased during storage at a(w) values of 0.89, 0.62 and 0.36 but there was little loss of viability in gelatin at an aw of 0.25 over 27 d storage at 26 degrees C. Assays for enterotoxin A detected no synthesis of new toxin but no loss in pre-formed toxin. The results suggest that high levels of Staph. aureus and its toxins should be excluded from glassy and rubbery state food products in order to prevent illness.

Colony Count, Microbial↗
PubMed1995

Specific antibody-mediated detection of Brochothrix thermosphacta in situ in British fresh sausage.

A rabbit polyclonal antibody-linked probe was developed which detected 76% of 800 food isolates of the spoilage bacterium Brochothrix thermosphacta when cells were bound to nitrocellulose. In slide cross-reaction tests all six environmental isolates tested were stained but the type strain was not. The antibody did not cross-react with Listeria grayi, L. monocytogenes, Lactobacillus plantarum, Lactococcus lactis, Streptococcus mutans, Bacillus cereus or B. subtilis. The antibody-linked probe detected Br. thermosphacta in thin sections of British fresh sausage when the viable count was greater than 10(6) g-1. Cells were detected mainly within 1 or 2 mm of the surface on the loose starchy material. They were not detected within muscle blocks or in the centre of the sausage. Such results suggest that growth of this organism occurs close to the surface of the sausage.

3,3'-Diaminobenzidine↗
PubMed1995

Locating nisin-producing Lactococcus lactis in a fermented meat system.

Antibody-linked probes were used to locate nisin in a fermented meat system. Free nisin or nisin bound to susceptible cells or food components was not detected. Colonies of nisin-producing Lactococcus lactis were stained at all times during growth. The position of nisin-producing L. lactis colonies was noted and compared with the location of spoilage organisms or the distribution of areas with a fermented meat appearance. No relationship between the distribution of starter culture and the location of spoilage organisms or areas of fermentation was observed. In addition to the presence of L. lactis, a rapidly fermentable sugar was also required to obtain a fermented appearance and to reduce the levels of spoilage organisms.

Animals↗
PubMed1994

Survival of Salmonella senftenberg and Salmonella typhimurium in glassy and rubbery states of gelatin.

Salmonella senftenberg and Salmonella typhimurium survived the production of gelatin sheets containing nutrient broth although there was some evidence of cell damage. Both strains survived but did not grow in glassy states with an a(w) of 0.45-0.28 and rubbery states with an a(w) of 0.93-0.96 for at least 28 d. Survival was less in intermediate states with an a(w) between 0.55 and 0.74. The results suggest that salmonellas should be excluded from glassy state products in order to prevent salmonellosis.

Colony Count, Microbial↗
PubMed1994

Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from being killed by freeze-drying.

Wild-type spores of Bacillus subtilis were resistant to eight cycles of freeze-drying, whereas about 90% of spores lacking the two major DNA-binding proteins (small, acid-soluble proteins alpha and beta) were killed by three to four cycles of freeze-dryings, with significant mutagenesis and DNA damage accompanying the killing. This role for alpha/beta-type small, acid-soluble proteins in spore resistance to freeze-drying may be important in spore survival in the environment.

Bacillus subtilis↗
PubMed1993

The application of laser diffractometry to study the water content of spores of Bacillus sphaericus with different heat resistances.

The distribution of water in the protoplast and integument of three populations of Bacillus sphaericus spores was determined by laser diffractometry together with the sizes of their integuments and protoplasts. The spores were produced at 15, 20 and 30 degrees C. Spores grown at 15 degrees C had protoplast and integument water contents similar to those produced at 20 degrees C, while those grown at 30 degrees C had significantly lower water contents than the other two populations. The inner (or protoplast) radii of the spores produced at 15, 20 and 30 degrees C were 0.41 +/- 0.02 microns, 0.42 +/- 0.01 microns and 0.38 +/- 0.02 microns whilst the outer (or whole spore) radii were 0.56 +/- 0.01 microns, 0.58 +/- 0.01 microns and 0.52 +/- 0.02 microns respectively. The ratios of integument to protoplast radius were 0.40 +/- 0.02, 0.43 +/- 0.07 and 0.41 +/- 0.03 respectively.

Bacillus↗