Evaluation of TA10 Broth for Recovery of Listeria monocytogenes from Ground Beef.

In 2009, the enrichment broth TA10 was released for simultaneous recovery of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. This medium was compared with other Salmonella enrichment broths [lactose (LAC) broth, buffered peptone water (BPW), and universal pre-enrichment (UP) broth] for the recovery of heat- and freeze-injured Salmonella spp. in beef by the conventional culture method. There was a significant difference between TA10 and LAC enrichment broths for detecting injured Salmonella spp. In this study, the International Organization for Standardization Listeria pre-enrichment broth (Half-Fraser/Fraser) was compared with TA10 broth for the recovery of L. monocytogenes from ground beef. Ground beef samples were contaminated with single Listeria serovars at levels of 0.096 to 0.001 most probable number/g. Twenty 25 g test portions of the contaminated ground beef were pre-enriched in each broth, and the ISO-11290-1 Listeria official isolation protocol was used thereafter. There was a significant difference between TA10 broth (48 h) and Half-Fraser/Fraser broth (72 h) in the recovery of L. monocytogenes. In addition, the incubation time for TA10 broth was shorter than for Half-Fraser/Fraser broth. The results indicate that TA10 broth should be used instead of Half-Fraser/Fraser broth for analysis of beef that may be contaminated with very low levels of L. monocytogenes.

Evaluation of TA10 broth for recovery of heat- and freeze-injured Salmonella from beef.

The Bacteriological Analytical Manual (BAM) Salmonella pre-enrichment broth [lactose (LAC) broth], buffered peptone water, and universal pre-enrichment (UP) broth were compared with TA10 broth, developed in our laboratory, for recovery of heat- and freeze-injured Salmonella (55 degrees C for 2-20 min and -20 degrees C for 2 months, respectively) from beef. Beef samples were contaminated with single Salmonella serovars, and contamination levels of 0.44 to <0.001 most probable number (MPN)/g and 0.74 to 0.14 MPN/g were used for heat- and freezing-induced injury studies, respectively. Twenty test portions (25 g) of the contaminated beef were pre-enriched in each broth, and the BAM Salmonella culture method was used thereafter. There was a significant difference (chi2 = 7.73) in recovery of heat-injured Salmonella between TA10 broth and LAC broth, 189 (67.5%) versus 156 (55.7%) positive samples, respectively, determined by plating onto selective agars and identification by biochemical tests. For the recovery of freeze-injured Salmonella, there was a significant difference (chi2 = 24.7) between TA10 and LAC broth, 189 (72.7%) versus 133 (51.2%) positive samples, respectively. TA10 broth was more effective than LAC broth and UP broth for recovery of freeze-injured Salmonella. The results indicate that TA10 broth should be used instead of LAC broth for testing of beef that may be contaminated with heat- and freeze-injured Salmonella spp.

Multiplex real-time polymerase chain reaction assay for simultaneous detection and quantification of Salmonella species, Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples.

Salmonella sp., Listeria monocytogenes, and Escherichia coli O157:H7 are foodborne pathogens capable of causing serious gastrointestinal illness. We previously described simultaneous detection of these pathogens by multiplex polymerase chain reaction (PCR) in 44 types of spiked food samples, including meat, produce, fish, and dairy products, targeting genes specific for each pathogen. Based on the previous work, a multiplex real-time PCR assay using fluorescent probes was developed to detect and accurately quantify Salmonella sp., L. monocytogenes, and E. coli O157:H7 in ground pork samples. The detection sensitivity for this method was 2.0 x 10(2) CFU/mL for each pathogen, and the quantification range was 10(2)-10(7) CFU/mL with a high correlation coefficient (R(2) > 0.99) and high PCR efficiency (84.2% to 99.2%). When this protocol was used for the detection of each of the pathogens in spiked pork samples, one cell per 25 g of inoculated sample after enrichment for 20 h could be detected within 24 h. As a result, this multiplex real-time PCR assay will be valuable as a screening method for foods contaminated with these pathogens.

Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing.

Conventional culture methods were compared to a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 from enrichment cultures of various types of artificially inoculated and naturally contaminated foods. The multiplex PCR assay was evaluated in 44 types of spiked food samples, including meat, produce, fish, and dairy products targeting genes specific for each pathogen for simultaneous detection. The sensitivity of the assay was

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples.

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.

Damage of yeast cells induced by pulsed light irradiation.

DNA damage, such as formation of single strand breaks and pyrimidine dimers was induced in yeast cells after irradiation by pulsed light, which were essentially the same as observed with continuous ultraviolet (UV) light. The UV-induced DNA damage is slightly higher than seen with pulsed light. However, increased concentration of eluted protein and structural change in the irradiated yeast cells were observed only in the case of pulsed light. A difference in the inactivation effect between pulsed light and UV light was found and this suggested cell membrane damage induced by pulsed light irradiation. It is proposed that pulsed light can be used as an effective sterilizing method for the yeast Saccharomyces cerevisiae.

Biochemical and genetic characterization of mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393.

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.