Mechanical damage to Gram-negative bacteria by surface plating with the Drigalski-spatula technique

Colony counting by spreading bacterial suspensions on plating media by various techniques is of general concern. Comparative studies between hand plating (Drigalski-spatula technique) for different time intervals and spiral plating resulted in significant differences in colony counts. Lower counts of Gram-negative bacteria were obtained by using hand plating for more than 10 s, compared with short time hand plating (5 s) or spiral plating. Colony counting of Gram-positive bacteria showed no differences between both techniques. Further characterisation of Escherichia coli cells spread with the Drigalski-spatula technique by electron microscopy revealed a large number of damaged cells compared to control samples. The data clearly shows that the mechanical forces during hand plating are sufficient to damage E. coli cells.     

Evaluation of standard and new chromogenic selective plating media for isolation and identification of Bacillus cereus

In this study, the performance of two new chromogenic plating media (CBC and BCM) was compared with two standard selective plating media (PEMBA and MYP) recommended by food authorities for isolation, identification and enumeration of Bacillus cereus. The four media types were challenged with a strain set comprising 100 B. cereus isolates from different origins and with different toxigenic potentials (40 food isolates, 40 isolates from food borne outbreaks and 20 clinical isolates). Additionally, the performance of the plating media for analysis of complex samples was assessed using naturally contaminated foods. Our survey showed that the new chromogenic media represent a good alternative to the conventional standard media. Especially, if laboratory staff are not highly trained in identification of B. cereus, the conventional media could lead to substantial misidentification and underestimation of food borne illness caused by B. cereus. However, there are some B. cereus strains that could not even be detected with this new type of chromogenic media. After the fatal misidentification of a highly toxic strain, other methods for a conclusive identification of B. cereus are needed. Sequence analysis of the plcR gene, a pleiotropic regulator of various virulence factors and B. cereus specific enzymes, revealed a significant correlation between atypical colony appearance and specific variances within the plcR gene sequences of those strains. The current concept of selective plating media, utilising PlcR regulated enzyme activities for differentiation purposes, should therefore be reconsidered and research should be geared towards culture independent methods.     

Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature

A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in Aspergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of a_w and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B₁ was low. At all other combinations (25 °C/0.95 and 0.99; 30 °C/0.95 and 0.99; 35 °C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis.     

Polycyclic aromatic hydrocarbons in human milk of nonsmoking U.S. women

The determination of polycyclic aromatic hydrocarbons (PAHs) in human milk is relevant in assessing the dose to mothers and infants. Since measurements for the United States are lacking, we analyzed 13 PAHs in human milk from 12 nonsmoking American mothers. Analytes were extracted from milk by using a combination of centrifugation, liquid-liquid, and solid-phase extraction techniques. Following method validation with spiked bovine milk, PAH concentrations in human milk were determined by isotope dilution gas chromatography/mass spectrometry. Phenanthrene, fluorene, fluoranthene, and pyrene were found at median concentrations of 11.8, 3.0, 1.6, and 1.1 ng/g milk fat, respectively, whereas 9 additional analytes including benzo[a]pyrene were not detected (<0.04 ng/g). Calculated average daily doses from ingestion (high: 75.8, phenanthrene; low: 6.1 ng/kg/day pyrene) were compared to doses from air pollution inhalation. Milk ingestion accounted for 47.9-81.4% of the total dose of individual PAHs to the infant. However, calculated total average daily doses of phenanthrene and pyrene from ingestion and inhalation combined (12.2 to 110.3 ng/kg-day, respectively) were 3 orders of magnitude lower than documented doses known to cause health effects. These first measurements of PAH levels in U.S. human milk are comparable to concentrations reported previously for European and Asian women.     

Use of nisin-coated plastic films to control Listeria monocytogenes on vacuum-packaged cold-smoked salmon

Cold-smoked (Salmo salar) salmon samples were surface-inoculated with a cocktail of three nisin-resistant strains of L. monocytogenes (PSU1, PSU2 and PSU21) to a level of approximately 5 x 10(2) or 5 x 10(5) CFU/cm2 of salmon surface. The inoculated smoked salmon samples were vacuum-packaged with control film (no nisin) or nisin-coated plastic films and stored at either 4 or 10 degrees C. When the inoculated smoked salmon samples were packaged with film coated with 2000 IU/cm2 of nisin, a reduction of 3.9 log CFU/cm2 (compared with control) was achieved at either temperature for samples inoculated with 5 x 10(2) CFU/cm(2 of L. monocytogenes after 56 (4 degrees C) and 49 (10 degrees C) days of storage while reductions of 2.4 and 0.7 log CFU/cm2 were achieved for samples inoculated with a high level of L. monocytogenes (5 x 10(5) CFU/cm2) after 58 (4 degrees C) and 43 (10 degrees C) days, respectively. For samples packaged in film coated with 500 IU/cm2 of nisin, reductions of 0.5 and 1.7 log CFU/cm2 were achieved for samples inoculated with a low level of L. monocytogenes (5 x 10(2) CFU/cm2) after 56 (4 degrees C) and 49 (10 degrees C) days of storage while reductions of 1.8 and 0.8 log CFU/cm2 were achieved for samples inoculated with high level of L. monocytogenes after 58(4 degrees C) and 43 (10 degrees C) days, respectively. In addition, nisin inhibited the proliferation of background microbiota on smoked salmon in a concentration-dependent manner at both storage temperatures although the bacteriostatic effect was more pronounced at refrigeration temperature. This work highlights the potential for incorporating nisin into plastic films for enhancing the microbial safety of smoked salmon as well as controlling its microbial spoilage.     

The influence of 1B/1R chromosome translocation on gluten protein composition and technological properties of bread wheat

The Austrian bread wheat Amadeus without and with 1BL/1RS translocation and three further translocation genotypes with known HMW subunit compositions were grown under the same environmental conditions. Their flours were characterised by the determination of crude protein content and, partly, by the determination of glutathione and cysteine. Furthermore, the qualitative and quantitative composition of gluten protein types was analysed by a combined extraction and reversed phase HPLC procedure. Dough development time, maximum resistance and extensibility of dough and gluten, and bread volume were determined by means of microscale methods. Protein, glutathione and cysteine contents of flours were only slightly influenced by translocation. The HPLC patterns of gliadins and glutenin subunits showed that translocation caused characteristic changes concerning ω‐gliadins, γ‐gliadins and LMW subunits of glutenin. The amount of ω 1,2‐gliadins was significantly increased and that of LMW subunits decreased. The effect of translocation on the rheological properties of dough and gluten was characterised by a strongly reduced dough development time, reduced maximum resistance and increased extensibility. Bread volume was decreased by about 10%. The amount of glutenin subunits was correlated with dough development time, resistance of dough and gluten, and bread volume to a higher extent (r = 0.79–0.91) than the amount of gliadins (r = 0.52–0.80). Correlation coefficients for LMW subunits were higher (r = 0.82–0.88) than those for HMW subunits (r = 0.35–0.61) when all five wheats were included. Instead, when only translocation lines were considered, HMW subunits (r = 0.89–0.98) were more important than LMW subunits (r = 0.64–0.86). Altogether, the results demonstrate that translocation causes important quantitative as well as qualitative changes in gluten protein composition which can be efficiently determined by reversed phase HPLC. © 2000 Society of Chemical Industry     

Influence of light on food relevant fungi with emphasis on ochratoxin producing species

The influence of light of varying wavelength on growth and ochratoxin A biosynthesis of Aspergillus carbonarius, A. niger, A. steynii and on Penicillium nordicum and P. verrucosum was analysed. For comparison the influence of light on various other food relevant fungi, including citrinin producers, was also tested. Generally the Aspergilli seem to be more resistant to light treatment than the Penicillia. Interestingly wavelengths from both sides of the spectrum, e. g. red (long wavelength, 627 nm) and blue (short wavelength 470-455 nm) had the strongest inhibitory effects on growth and ochratoxin A biosynthesis. Blue light generally had a stronger effect. Light of moderate wavelength, 590 to 530 nm, (yellow to green) had more a positive than a negative influence on growth or ochratoxin A biosynthesis compared to the control (dark incubation). The light effect on growth and ochratoxin A biosynthesis was dependent on the growth medium. In contrast to malt extract medium (MEA), YES medium, as an especially nutrient rich medium, had an attenuating effect on the reactivity against light. However the tendency of the response in both media was the same. Moreover, the light intensity strongly influences how the fungus reacts. Depending on the intensity and the resistance of the species a complete cessation of growth and/or inhibition of ochratoxin A biosynthesis could be achieved. Light irradiation has the opposite effect on ochratoxin A than citrinin, two mycotoxins which can be produced simultaneously in P. verrucosum. Citrinin was produced essentially under light conditions which inhibited ochratoxin A biosynthesis. The same was true for a derivative of ochratoxin, in particular a derivative of ochratoxin β in A. carbonarius. A. carbonarius produced high amounts of the ochratoxin β derivative under blue light when the production of ochratoxin A was ceased at the most inhibiting conditions used (MEA, royal blue light, 455 nm, 1700 lx). Light has a growth stalling but not inactivating effect on aerial mycelia. If a non-growing colony under light is shifted to the dark it immediately grows normally. However on spores blue light has a deactivating effect. After incubation of spores of P. verrucosum for 24 h under blue light up to 97% of the spores were no longer able to germinate. Again the spores of the Aspergilli were much more resistant.     

Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia coil O157:H7, Salmonella,and Listeria with BAX,and evaluation of this polymerase chain reaction-based system with experimentally contaminated samples

BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.     

Shock Consolidation of Aluminium Powder with SiC fiber reinforcement

For the explosive compaction of Al 2024 alloy powder the influence of E/M ratio and detonation velocity of the explosive as well as the influences of powder microhardness and initial density are discussed. With a choice of parameters that gives a fully compacted aluminium sample the results of embedding single SiC fibers are described and a procedure to avoid fiber cracking due to shock loads is derived. First results for the compaction of samples with 38% fiber content are presented.