Development and application of new nucleic acid-based technologies for microbial community analyses in foods

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.     

Victoria＇s Secret——用商品目录卖服装

Victorias Secret乱是一家由女性经营，井专为女性服务的美国公司,为了要不断地站在时尚前端，每隔五六年，Victorias Secret所有的店面都会进行改版，以追求让人愿意进来花钞票换一件比牛仔裤还要贵的内衣产品。     

Dietary fibre content of thirteen apple cultivars

Fibre composition of the following 13 apple cultivars was studied: ‘Cortland’, ‘Empire’, ‘Fuji’, ‘Golden Delicious’, ‘Gala’, ‘Granny Smith’, ‘Jonagold’, ‘Mutsu’, ‘McIntosh’, ‘Delicious’, ‘Rome’, ‘Stayman’ and ‘York’. Fruit samples from each of these cultivars were analysed for non-starch cell wall materials (NSCWM) and non-starch polysaccharides (NSP). NSCWM was further fractionated into soluble and insoluble fibre fractions. Both NSCWM and NSP content were found to be significantly influenced by cultivar. NSCWM content ranged from 19·1 g kg⁻¹ apple flesh in ‘Fuji’ to 36·2 g kg⁻¹ in ‘York’. Mean(±SD) NSCWM content of all the cultivars was 23·1±4·5 g kg⁻¹. NSP content of apple flesh ranged from 13·8 g kg⁻¹ in ‘McIntosh’ to 28·7 g kg⁻¹ in ‘York’ with the overall mean for all cultivars being 17·9±4·2 g kg⁻¹. Relative amount of monosaccharides found in the hydrolysates of apple fibre also varied among cultivars. The greatest difference was observed in galactose content. ©1997 SCI     

Growth inhibition of Escherichia coli O157:H7 and Listeria monocytogenes by carvacrol and eugenol encapsulated in surfactant micelles

Growth inhibition of four strains of Escherichia coli O157:H7 (H1730, F4546, 932, and E0019) and Listeria monocytogenes (Scott A, 101, 108, and 310) by essential oil components (carvacrol and eugenol) solubilized in nonionic surfactant micelles (Surfynol 465 and 485W) was investigated. Concentrations of encapsulated essential oil components ranged from 0.02 to 1.25% depending on compound, surfactant type, and surfactant concentration (0.5 to 5%). Eugenol encapsulated in Surfynol 485W micelles was most efficient in inhibiting growth of the pathogens; 1% Surfynol 485W and 0.15% eugenol was sufficient to inhibit growth of all strains of E. coli O157:H7 and three of four strains of L. monocytogenes (Scott A, 310, and 108). The fourth strain, L. monocytogenes 101, was inhibited by 2.5% Surfynol and 0.225% eugenol. One percent Surfynol 485W in combination with 0.025% carvacrol was effective in inhibiting three of four strains of E. coli O157:H7. Strain H1730 was the most resistant strain, requiring 0.3% carvacrol and 5% surfactant for complete inhibition. Growth inhibition of L. monocytogenes by combinations of carvacrol and Surfynol 465 ranged between 0.15 and 0.35% and 1 and 3.75%, respectively. Generally, the antimicrobial activity of Surfynol 465 in combination with eugenol was higher than that for the combination with carvacrol. The potent activity was attributed to increased solubility of essential oil components in the aqueous phase due to the presence of surfactants and improved interactions of antimicrobials with microorganisms.     

Automated high-throughput immunomagnetic separation-PCR for detection of Mycobacterium avium subsp. paratuberculosis in bovine milk

Two monoclonal antibody-mediated immunomagnetic separation PCR kits (AnDiaTec ParaTub-S® IMS-PCR-ELISA and ParaTub-SL® IMS-real time PCR) were developed and evaluated for the automated high-throughput detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in bulk milk of naturally infected dairy herds and made commercially available. M. paratuberculosis are first isolated from milk by high-throughput immunomagnetic bead separation using a completely automated magnetic particle pipetting robot within 45 min and released subsequently for analysis directly into PCR amplification mixtures for real time PCR or for PCR-ELISA. The threshold detection level and specificity of the tests were evaluated first with different M. paratuberculosis pure cultures and artificially contaminated (spiked) bulk milk samples. Both experiments proved a good detection limit, specificity and reliability of the tests that consistently detected 20 or less M. paratuberculosis organisms from cattle, deer and mufflon in 1 ml milk. Experiments with more than 200 bulk milk samples that were tested in parallel with the PCR methods and with the cultural method in a second evaluation study demonstrated that both PCR tests are superior to culture and sufficiently sensitive to detect single shedders in pooled milk samples. The experiments proved that the newly developed tests are sensitive, specific and fast, and thus for the first time allow the standardized large-scale routine M. paratuberculosis screening of bulk milk samples at acceptable costs.     

Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada

This study determined the prevalence of Salmonella serovars, antimicrobial resistance (AMR) and resistance genes in Salmonella isolated from retail meats purchased in Alberta, Canada. Samples were collected during one year period (May 2007–April 2008) on weekly basis from 19 census divisions in Alberta. A total of 564 samples including chicken (n = 206), turkey (n = 91), beef (n = 134) and pork (n = 133) were purchased. Salmonella were recovered from chicken (40%), turkey (27%) and pork (2%) samples and was not found in ground beef. A total of 21, 8, and 3 different serovars were recovered from chicken, turkey and pork meats, respectively. Salmonella Hadar was most common in chicken whereas S. Heidelberg was common in turkey meat. Overall 29% (32/110) of isolates were susceptible to tested antimicrobials and resistance to ciprofloxacin, amikacin and nalidixic acid was not found in any isolate. Multiresistance (≥2 antimicrobials) was found in 56% of isolates. Resistance to amoxicillin–clavulanic acid (AMC), ceftiofur (TIO), and ceftriaxone (CRO) was found in about 21% of chicken and 25% of turkey isolates. Resistance to either of tetracycline (TET), streptomycin (STR) or ampicillin (AMP) was unconditionally associated with S. Hadar but resistance to either of TET, AMP, AMC, TIO, CRO or cefoxitin was associated with S. Heidelberg. The strA/B (42% isolates), tet(A) (28% isolates), bla_CMY-2 (21% isolates) and bla_TEM (17% isolates) were the most common resistance genes found. The bla_CMY-2 and bla_TEM genes were unconditionally associated with S. Heidelberg; tet(A) and strA/B with S. Hadar and tet(B) gene with S. Kentucky. The strA/B genes were not associated with S. Heidelberg. Our data suggests that the prevalence of Salmonella serovars varied by the meat type and that AMR and resistance genes varied by the Salmonella serovars.