Simultaneous Identification of 13 Foodborne Pathogens by Using Capillary Electrophoresis-Single Strand Conformation Polymorphism Coupled with Multiplex Ligation-Dependent Probe Amplification and Its Application in Foods.

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with stuffer-free multiplex ligation-dependent probe amplification (MLPA) was developed to identify 13 species of foodborne pathogens simultaneously. Species-specific MLPA probes were designed for nine of these species. These probes were targeted to the groEL, glyA, MMS, tuf, inv, ipaH, nuc, vvh, and 16S rRNA genes, which corresponded to Bacillus cereus, Campylobacter coli, Cronobacter sakazakii, Enterococcus spp., Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio vulnificus, and Yersinia enterocolitica, respectively. MLPA probes that had been previously developed by our laboratory were used for the other four species (Campylobacter jejuni, Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes). The CE-SSCP method was optimized to identify all 13 foodborne microbes simultaneously in a single electrogram, in which 50-500 pg genomic DNA was detected per microbe. Twelve species were detected from animal-derived food samples (specifically, milk and sliced ham) that had been artificially inoculated with 12 of the foodborne pathogens, excluding V. vulnificus, which is not usually associated with animal foods. The method developed here could be used as an early warning system for outbreaks of foodborne diseases associated with animal-derived foods in the food industry.

WNT Pathway Mutations in Metachronous Oligometastatic Castration-Sensitive Prostate Cancer.

PURPOSE: WNT signaling is a cellular pathway that has been implicated in the development and progression of prostate cancer. Oligometastatic castration-sensitive prostate cancer (omCSPC) represents a unique state of disease in which metastasis-directed therapy (MDT) has demonstrated improvement in progression-free survival. Herein, we investigate the clinical implications of genomic alterations in the WNT signaling cascade in men with omCSPC. METHODS AND MATERIALS: We performed an international multi-institutional retrospective study of 277 men with metachronous omCSPC who underwent targeted DNA sequencing of their primary/metastatic tumor. Patients were classified by presence or absence of pathogenic WNT pathway mutations (in the genes APC, RNF43, and CTNNB1). Pearson χ2 and Mann-Whitney U tests were used to determine differences in clinical factors between genomic strata. Kaplan-Meier survival curves were generated for radiographic progression-free survival and overall survival, stratified according to WNT pathway mutation status. RESULTS: A pathogenic WNT pathway mutation was detected in 11.2% of patients. Patients with WNT pathway mutations were more likely to have visceral metastases (22.6% vs 2.8%; P < .01) and less likely to have regional lymph node metastases (29.0% vs 50.4%; P = .02). At time of oligometastasis, these patients were treated with MDT alone (33.9%), MDT + limited course of systemic therapy (20.6%), systemic therapy alone (22.4%), or observation (defined as no treatment for ≥6 months after metastatic diagnosis). Multivariable cox regression demonstrated WNT pathway mutations associated with significantly worse overall survival (hazard ratio, 3.87; 95% confidence interval, 1.25-12.00). CONCLUSIONS: Somatic WNT pathway alterations are present in approximately 11% of patients with omCSPC and are associated with an increased likelihood of visceral metastases. Although these patients have a worse natural history, they may benefit from MDT.

Synergistic effect of sous-vide and fruit-extracted enzymes on pork tenderization.

A combination of sous-vide (SV) and enzymatic treatment (one commercial, Neutrase (NE), and two fruit-extracted enzymes obtained from kiwifruit and pineapple, KE and PE, respectively) was applied to pork fore shanks prepared at different temperatures (45, 60, 70, and 100 °C) for 0.5, 4 or 8 h, and the properties of pork were compared. For the hardness, SV itself resulted in a 27% decrease; however, a significant softening effect could be obtained by the addition of enzymes (38-60%). The KE treatment appeared to be more effective (~ 60%) than either the PE or the NE treatment. During SV, both the L* and b* values of the samples generally increased while the a* value decreased. Among the samples, the lowest hardness was obtained for the sample treated with SV-KE at 70 °C for 8 h, and the lowest total microbial count, lowest pH and the least amount of color change were also observed for the sample.

Expression of a recombinant Cry1Ac crystal protein fused with a green fluorescent protein in Bacillus thuringiensis subsp. kurstaki Cry-B.

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis Cry(-)B strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the cry1Ac gene (pProAc-GFP). The B. thuringiensis Cry(-)B strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immunoblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single species, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis Cry(-)B strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuringiensis.

Effect of Lactobacillus acidophilus KFRI342 on the development of chemically induced precancerous growths in the rat colon.

Lactobacillus acidophilus KFRI342, isolated from the Korean traditional food kimchi, was investigated for its suitability as a dietary probiotic. The effects of L. acidophilus KFRI342 on the development of chemically induced (1,2-dimethylhydrazine; DMH) precancerous cytological changes of the colon were investigated in rats. Forty-five male F344 rats were randomly divided into three dietary groups. The control group received a high-fat diet (HF), a second group received a high-fat diet containing the carcinogen (HFC), and a final group received a high-fat diet containing the carcinogen and L. acidophilus KFRI342 (HFCL). L. acidophilus KFRI342 was administered orally three times per week at 2×10(9) c.f.u. ml(-1). L. acidophilus KFRI342 treatments decreased the number of Escherichia coli in faecal samples, the enzyme activities of ß-glucuronidase and ß-glucosidase, and plasma triglyceride concentration compared to the HF and HFC treatments (P<0.05). L. acidophilus KFRI342 consumption also decreased the ratio of aberrant crypts to aberrant crypt foci incidence and the number of aberrant crypts in HFCL rats. Therefore, L. acidophilus showed potential probiotic activity as an inhibitor of DMH-induced symptoms in live rats. Our in vivo studies indicate that L. acidophilus from kimchi may be suitable as a probiotic for human use.

[Association between prophylactic antibiotic use and surgical site infection based on quality assessment data in Korea].

OBJECTIVES: To examine the prophylactic antibiotic use in reducing surgical site infection. METHODS: This was a retrospective study for patients aged 18 years and older who underwent gastrectomy, cholecystectomy, colectomy, cesarean section and hysterectomy. The data source was quality assessment data of the Health Insurance Review & Assessment Service gathered from medical records of 302 national hospitals. Prophylactic antibiotic use was defined as: timely antibiotic administration or inappropriate antibiotic selection. We performed hierarchical logistic regression to examine the association between prophylactic antibiotic use and surgical site infection with adjustment for covariates. RESULTS: The study population consisted of 16, 348 patients (1,588 gastrectomies, 2,327 cholecystectomies, 1,384 colectomies, 3,977 hysterectomies and 7,072 cesarean sections) and surgical site infection was identified in 351 (2.1%) patients. The rates of timely antibiotic administration and inappropriate antibiotic selection varied according to procedures. Cholecystectomy patients who received timely prophylactic antibiotic had a significantly reduced risk of surgical site infection compared with those who did not receive a timely prophylactic antibiotics (OR 0.64, 95% CI=0.50-0.83), but no significant reduction was observed for other procedures. When inappropriate prophylactic antibiotics were given, the risk of surgical site infection significantly increased: 8.26-fold (95% CI=4.34-15.7) for gastrectomy, 4.73-fold (95% CI=2.09-10.7) for colectomy, 2.34-fold (95% CI=1.14-4.80) for cesarean section, 4.03-fold (95% CI=1.93-8.42) for hysterectomy. CONCLUSIONS: This study examines the association among timely antibiotic administration, inappropriate antibiotic selection and surgical site infection. Patients who received timely and appropriate antibiotics had a decreased risk of surgical site infection. Efforts to improve the timing of antibiotic administration and use of appropriate antibiotic are needed to lower the risk of surgical site infection.

An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin.

Baculovirus occlusion bodies, large proteinaceous structures which contain virions, have recently been engineered to incorporate foreign proteins. The major constituent protein of occlusion bodies from the baculovirus Autographa californica nucleopolyhedrovirus is polyhedrin, and assembly of recombinant occlusion bodies which incorporate a foreign protein depends on an interaction between native polyhedrin and a polyhedrin-foreign protein fusion. This technology has now been applied to the generation of a recombinant baculovirus (ColorBtrus) that produces occlusion bodies incorporating the Bacillus thuringiensis (Bt) insecticidal Cry1Ac toxin protein. ColorBtrus coexpresses native polyhedrin and a fusion protein in which polyhedrin is fused to the Bt toxin, which is in turn fused to green fluorescent protein (GFP). Analysis of ColorBtrus occlusion bodies confirmed that they include both Bt toxin and GFP, yet still incorporate virions. Bioassay of ColorBtrus demonstrated that its speed of action and pathogenicity are strikingly enhanced compared to wild-type virus. ColorBtrus represents a novel, powerful biological insecticide that combines positive attributes of both Bt toxin and baculovirus based systems.

Isolation and characterization of a strain of Bacillus thuringiensis subsp. morrisoni PG-14 encoding delta-endotoxin Cry1Ac.

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.