Horizontal transmission of antimicrobial resistance genes from E. coli to Serratia spp. in minced meat using a gfp tagged plasmid.

The transmission of antimicrobial resistance genes from enteric bacteria from the animal reservoir to indigenous bacteria in meat is a serious concern, as it can contribute to human exposure to antimicrobial resistance genes. The aim of this study was to investigate plasmid-mediated horizontal transfer of antimicrobial resistance genes from Escherichia coli to indigenous environmental bacteria in minced pork stored at 10 and 37 °C. E. coli MG1555 containing a gfp-tagged plasmid carrying tetracycline, kanamycin and streptomycin resistance genes was used as the donor with the indigenous bacteria in minced pork acting as potential recipients. The results demonstrated that enteric members of the pork meat microbiota were able to receive gfp-plasmids from the E. coli donor strain at both 10 and 37 °C. The majority of transconjugants were identified as Serratia spp. through sequencing of their 16S rRNA genes. This indicates that environmental Serratia spp. and other Enterobacteriaceae may play a role as carrier of antimicrobial resistance genes through the meat production chain to the consumer.

Cross and co resistance among Danish porcine E. coli isolates.

Cross and co-resistance to antimicrobials are presented for 765 Danish Escherichia coli isolates of porcine origin from 2009 to 2013. All isolates and data originate from the DANMAP surveillance but have not previously been used to describe the occurrence of cross and co- resistance. Data presented here clearly indicate the ability of low classified antimicrobials as ampicillin to uphold resistance to critical important antimicrobials for human treatment.

Comparative Investigation of Postoperative Complications in Patients With Gastroesophageal Junction Cancer Treated With Preoperative Chemotherapy or Surgery Alone.

BACKGROUND AND AIM: Gastroesophageal junction cancer is one of the leading causes to cancer-related death and the prognosis is poor. However, progress has been made over the last couple of decades with the introduction of multimodality treatment and optimized surgery. Three-year survival rates have improved to 50% in patients receiving neoadjuvant therapy. Only a few studies have focused on the difference of postoperative complications in patients receiving neoadjuvant therapy in relation to a comparative surgery-only group. The aim of this study was to compare the prevalence of postoperative complications of patients with cancer at the gastroesophageal junction treated with either neoadjuvant chemotherapy or surgery alone in patients from "The Danish Clinical Registry of Carcinomas of the Esophagus, the Gastro-Esophageal Junction and the Stomach." MATERIALS AND METHODS: A historical follow-up study, comparing postoperative complications between two cohorts before and after implementation of chemotherapy was completed. RESULTS: In all, 180 consecutive patients treated with perioperative chemotherapy and a comparative surgery-only group of patients were identified from The Danish Clinical Registry of Carcinomas of the Esophagus, the Gastro-Esophageal Junction and the Stomach. No difference was found in demographics between the two groups, except for alcohol consumption and a lower T and N stage in the surgery-only group, and no difference in complication rates was found. Furthermore, no variable in the multivariate analysis was significantly associated with anastomotic leakage which was considered the most severe complication. CONCLUSION: Since perioperative chemotherapy does not appear to increase surgical complications, the future challenges include defining the optimal combination of chemo- and/or radiotherapy, but more importantly also to select the patients who will benefit the most from the different neoadjuvant strategies.

Investigating the underlying mechanisms of temperature-related skin diseases in Atlantic salmon, Salmo salar L., as measured by quantitative histology, skin transcriptomics and composition.

Skin integrity is recognized as of vital consideration for both animal welfare and final product quality of farmed fish. This study examines the effects of three different rearing temperatures (4, 10 and 16 °C) on the skin of healthy Atlantic salmon post-smolts. Changes in skin condition were assessed by the means of skin composition analyses, quantitative histology assessments and transcriptome analysis. Level of protein, vitamin C and vitamin E was significantly higher at 16 °C compared with 4 °C. Quantitative histology measurements showed that the epidermal thickness decreased from low to high temperature, whereas the epidermal area comprising mucous cells increased. The difference was only significant between 4 and 16 °C. Both high and low temperature exhibited significant changes in the skin transcriptome. A number of immune-related transcripts responded at both temperatures. Contrary to well-described immunosuppressive effects of low water temperature on systemic immunity, a subtle increase in skin-mediated immunity was observed, suggesting a pre-activation of the mucosal system at 4 °C. Upregulation of a number of heat-shock proteins correlating with a decrease in epidermal thickness suggested a stress response in the skin at high temperature. The results demonstrate distinctive temperature-related effects on the skin of Atlantic salmon.

Proteomic analysis of epidermal mucus from sea lice-infected Atlantic salmon, Salmo salar L.

Health diets that contain immunostimulants and other functional ingredients can strengthen the immune response in Atlantic salmon, Salmo salar, and thereby reduce sea lice, Lepeophtheirus salmonis, infection levels. Such diets can be used to supplement other treatments and will potentially reduce the need for delousing and medication. A sea lice infection trial was conducted on fish with an average weight of 215 g. One control diet and four experimental diets containing functional ingredients were produced. The diets were fed to salmon for 4 weeks before infection with sea lice copepodids. When lice had developed to chalimus III/IV, 88 fish per diet were examined for lice loads. Mucus samples from fish fed the different diets were taken before and after lice infection. Mass spectrometry-based proteomics was used to characterize the protein composition in the epidermal mucus of Atlantic salmon and to identify quantitative alterations in protein expression. Multivariate analysis of the generated data sets was performed to identify protein biomarkers. Putative biomarkers associated with functional feed intake and with sea lice infection have been identified and can form the basis for strategic validation experiments with selected functional feeds.

In vitro penetration properties of solid lipid nanoparticles in intact and barrier-impaired skin.

Treatment of skin diseases implies application of a drug to skin with an impaired epidermal barrier, which is likely to affect the penetration profile of the drug substance as well as the carrier into the skin. To elucidate this, the effect of skin barrier damage on the penetration profile of a corticosteroid applied in solid lipid nanoparticles (SLN) composed of different lipids, varying in polarity, was studied. The studies were carried out in vitro using impaired and intact porcine ear skin, and the SLN were compared with a conventional ointment. It was shown that a significantly higher amount of corticosteroid remained in the skin, intact as well as barrier impaired, when SLN was used as a vehicle. In general, the penetration profile of the drug substance into the skin was affected by the type of lipid used in the formulation and related to lipid polarity and drug substance solubility. When formulated in SLN and applied to intact skin, the permeation of the drug substance across the skin was significantly reduced, as compared to the ointment. Altogether, in both barrier-impaired and intact skin, a higher amount of drug substance remained in the skin during application of SLN for 6, 16, and 24h, as compared to the ointment. These results emphasize the applicability of SLN to create a drug reservoir in skin, with the drug localized distinctively in the stratum corneum.

Host plant suitability and feeding preferences of the grapevine pest Abagrotis orbis (Lepidoptera: Noctuidae).

Thirteen plant species were tested for their suitability as hosts for Abagrotis orbis (Grote), a climbing cutworm pest of grapevines in British Columbia. Choice tests were also conducted to investigate larval feeding preferences for the Brassicaceae species joi choi, Brassica rapa variety. Chinensis L., spring draba; Draba verna L.; and shepherd's purse, Capsella bursa-pastoris (L.) Medik; compared with postdormant buds of grape, Vitis vinifera L. (Vitaceae), and leaves of nine other plant species from several families. Results showed that tah tsai, Brassica rapa L. variety rosularis (M. Tsen & S. H. Lee) Hanelt (Brassicaceae), is a superior host for A. orbis based on shorter time to adult eclosion, heavier pupae, and higher rates of survival. Later-instar larvae died when fed draba, whereas those reared on shepherd's purse did not survive beyond the third instar. White clover, Trifolium repens L. (Fabaceae), and grape leaves were unsuitable hosts throughout development. Fifth-instar A. orbis preferred plants of the Brassicaceae family, dandelion, Taraxacum officinale Weber (Asteraceae), and strawberry, Fragaria sp. L. (Rosaceae), compared with postdormant grape buds. The results of this study suggest that the winter annual mustards draba and shepherd's purse that often grow abundantly in vine rows might help reduce climbing cutworm damage to the buds of grapevines.

A classification system for plasmids from enterococci and other Gram-positive bacteria.

A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating genes (rep), alignment of these sequences and using a cutoff value of 80% identity on both protein and DNA level, 19 replicon families (rep-families) were defined together with several unique sequences. The prevalence of these rep-families was tested on 79 enterococcal isolates from a collection of isolates of animal and human origin. Difference in prevalence of the designed rep-families were detected with rep(9) being most prevalent in Enterococcus faecalis and rep(2) in Enterococcus faecium. In 33% of the tested E. faecium and 32% of the tested E. faecalis no positive amplicons were detected. Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup.

Frequent amplifications and deletions of G1/S-phase transition genes, CCND1 and MYC in early breast cancers: a potential role in G1/S escape.

Uncontrolled growth of cancer cells can be related to dysfunctional cell cycle control, including entry into S-phase, initiating cell division. Cyclin CCND3 and CCNE1 along with CDK2 and CDK6 regulate this checkpoint, and genetic changes, detectable by fluorescence in situ hybridization, are hypothesized to increase the aggressiveness of breast cancer, thereby influencing patient survival. Genomic change was investigated in 106 primary breast cancer samples, where the combined gene copy number changes in one of these four cell cycle regulatory factors was observed in 22% of the 98 tumors of successful analysis, distributed with 15 deletions and 7 amplifications. A trend towards decreased survival was observed with the aberrations, suggesting a prognostic potential of this set of markers, which was supported by an association with tumor grade. For validation of the new set of FISH probes for the G1/S-phase cell cycle factors, two additional markers, frequently amplified in breast cancers, were included in this study: The G1/S phase control gene CCND1 and the proliferation marker MYC. Both markers were amplified in 14% and deleted in 5% of the cases. This is the first report of genomic deletions of MYC in breast cancer.

Peanut cross-reacting allergens in seeds and sprouts of a range of legumes.

BACKGROUND: Recently, peanut-allergic patients have reported symptoms upon ingestion of bean sprouts produced from various legumes. OBJECTIVE: This study was designed to identify immunoreactivity to seeds and sprouts of legumes other than peanut in sera from peanut-allergic patients. METHODS: Crude protein extracts of seeds and sprouts (comprising cotelydons and hypocotyls/epicotyls) of peanut, soybean, green pea, blue lupine, mung bean, alfalfa, broad bean, and azuki bean were prepared. The reactivity of sera from 10 peanut-allergic patients to these extracts was analysed by indirect histamine release (HR), enzyme-allergosorbent test (EAST), EAST inhibition, and Western blots. Skin prick tests (SPTs) were performed on the patients with fresh legume seeds as well as four commercial legume sprouts, and food challenges with soybean, pea, and lupine were performed on a subgroup of the patients. RESULTS: All legume seeds and commercial sprouts induced positive SPTs in some of the patients. Indirect HR experiments indicated an extensive co-reactivity between peanut and the legumes, and cross-reactivity was observed for soybean, pea, and lupine seeds as well as lupine hypocotyls in EAST inhibition experiments. Of the 16 protein extracts, soybean, pea, and lupine seed extracts produced visible bands in Western blots. CONCLUSION: The symptoms reported by peanut-allergic patients after legume sprout intake might be caused by cross-reactivity of peanut-specific antibodies. The intake of raw legume sprouts might cause symptoms in peanut-allergic patients.

Using data on resistance prevalence per sample in the surveillance of antimicrobial resistance.

OBJECTIVES: In most existing antimicrobial resistance monitoring programmes, one single bacterial colony from each collected sample is susceptibility tested against a panel of antimicrobials. Detecting the proportion of colonies resistant to different antimicrobials in each sample can provide quantitative data on antimicrobial resistance (resistance prevalence per sample). METHODS: In this study, a total of 98 faecal samples from slaughter pigs were tested for tetracycline and sulphonamide resistance in Escherichia coli using the single colony method, and these results were compared with the results obtained using the resistance prevalence per sample method. RESULTS: The results obtained by the resistance prevalence per sample method showed a lower occurrence of resistance. Tetracycline resistance in E. coli was found in 36.7% of the samples using the single colony method, while the mean tetracycline resistance prevalence was 22.5% using the resistance prevalence per sample method. Similarly, sulphonamide resistance was 32.7% using the single colony method and 19.6% when using the resistance prevalence per sample method. Although different estimates were obtained by each method, the correlation test and the regression model demonstrated that there is a significant association between the results obtained using both methods (P value <0.01) for both antimicrobials tested. CONCLUSIONS: To support risk assessment and analysis of the association between consumption of antimicrobials and occurrence of resistance, there is a need to move towards a more quantitative approach when dealing with antimicrobial resistance in a population, and the resistance prevalence per sample method can provide some of this additional information.

Genetic alterations of CCND1 and EMSY in breast cancers.

AIMS: CCND1 and EMSY, on 11q13, are frequently amplified in breast cancer. CCND1 is implicated in cell cycle progression and EMSY is a BRCA2-associated repressor protein. The aim was to investigate gene copy numbers of CCND1 and EMSY and to determine if CCND1 amplification is associated with reduced survival of tamoxifen-treated breast cancer patients. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) was performed on 111 consecutive and 354 oestrogen receptor (ER)+ tamoxifen-treated breast cancers. In the consecutive set, CCND1 and EMSY were amplified in 14.8% and 7.2%, respectively, and deleted in 8.7% and 13.5%, respectively. In the ER+ set, CCND1 and EMSY were amplified in 20.6% and 9.6%, respectively, and deleted in 1.7% and 4.2%, respectively. CCND1 and EMSY gene amplifications were associated with decreased overall survival (OS) (P = 0.03 and P = 0.04, respectively) of patients in the ER+ set. CONCLUSION: As hypothesized, CCND1 amplifications are associated with poor OS in ER+ patients. EMSY amplification is also associated with poor OS. However, as >70% of EMSY amplifications were CCND1 amplified, EMSY may not have any additional effect on survival of ER+ breast cancer.

A possible mechanism of macrolide resistance among multiple resistant Campylobacter jejuni and Campylobacter coli isolated from Thai children during 1991-2000.

A total of 495 Campylobacterjejuni and 122 C. coli isolated from Thai children were screened for macrolide (erythromycin and azithromycin) resistance by disk diffusion assay. Minimum inhibitory concentrations for erythromycin, azithromycin, nalidixic acid, ciprofloxacin, tetracycline, streptomycin, gentamicin and chloramphenicol were further determined for these macrolide-resistant Campylobacter isolates. Presence of known point mutations resulting in reduced susceptibility to macrolides was investigated by PCR and DNA sequencing. Seventeen percent (23/122) of C. coli and 2.4% (12/495) of C. jejuni isolates were resistant to macrolides. By sequencing domain V of the 23S ribosomal DNA from all 35 macrolide-resistant isolates, a known point mutation of 23S rRNA associated with reduced susceptibility to macrolides was detected in all isolates except one. Among the macrolide-resistant isolates, all were multiply resistant to nalidixic acid and ciprofloxacin, of which the latter is the preferred antimicrobial used for diarrheal treatment in Thailand. Furthermore, most macrolide-resistant isolates were also resistant to tetracycline and streptomycin. The spread of macrolide and quinolone resistant Campylobacter should be monitored closely in Thailand and elsewhere as these antimicrobials are preferred drugs for treatment of diarrhea.

Incidence and transmission of a granulovirus in a large codling moth [Cydia pomonella L. (Lepidoptera: Tortricidae)] rearing facility.

Incidences of potential per os Cydia pomonella granulovirus (CpGV) transmission within a large codling moth colony were identified. CpGV was detected in the water which is used to wash egg sheets. When pre-neonates were extracted from eggs prior to emergence and tested for the presence of CpGV, 40% were found to carry amounts of CpGV detectable by a polymerase chain reaction (PCR) assay, suggesting possible transovarial transmission of the virus. Although symptoms typical of virus infection and larval death were found infrequently within communal rearing trays, the frequency with which CpGV DNA was detected by PCR assays increased from a mean of 31% of 10-day-old larvae to 94% of 25-day-old larvae. CpGV in codling moth cadavers remained virulent after being held at 60 degrees C for 3 days under conditions similar to the treatment of spent diet at the rearing facility before its disposal. PCR tests of surface samples taken from air filters and rearing rooms of the rearing facility were found to contain CpGV. Bioassays of surface samples from the diet trash bin and a filter through which outside air is passed before entering the rearing chambers resulted in significant codling moth neonate mortality. The virulence of CpGV in dust from the spent diet and the original inadvertent positioning of the diet trash bin directly below one of the air intake ducts are suggested as a possible additional source of CpGV contamination within the facility.

Diarrhetic shellfish poisoning toxin esters in Danish blue mussels and surf clams.

Until recently, little focus was given to the presence of diarrhetic shellfish poisoning (DSP) toxin esters in seafood products. However, during the last few years, the occurrence of a high percentage of esters of the total amount of DSP toxins present in some seafood products has been observed. Samples of Danish surf clams (Spisola spp.) and blue mussels (Mytilus edulis) from 1999-2004 were analysed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the presence of DSP toxin esters. The samples contained only okadaic acid and esters of okadaic acid. The level of total okadaic acid equivalents ranged from 224 to 2516 microg kg-1 in surf clams. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 83 to 98%, mean 95%. The level of total okadaic acid equivalents ranged from 43 to 1631 microg kg-1 in blue mussels. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 21 to 86%, mean 59%. The probability of a high percentage of okadaic acid esters seems to increase with higher amounts of total okadaic acid equivalents in the bivalves. The large prevalence of DSP toxin esters are of particular importance because of the increased use of chemical methods instead of mouse bioassay for the detection of DSP toxicity.

Reactions of [NH3+*, H2O] with carbonyl compounds: a McLafferty rearrangement within a complex?

The reactions of the water solvated ammonia radical cation [NH(3)(+*), H(2)O] with a variety of aldehydes and ketones were investigated. The reactions observed differ from those of low energy aldehydes and ketones radical cations, although electron transfer from the keto compound to ionized ammonia is thermodynamically allowed within the terbody complexes initially formed. The main process yields an ammonia solvated enol with loss of water and an alkene. This process corresponds formally to a McLafferty fragmentation within a complex. With aldehydes, another reaction can take place, namely the transfer of the hydrogen from the CHO group to ammonia, leading to the proton bound dimer of ammonia and water, and to the NH(4)(+) cation. Comparison between the available experimental results leads to the conclusion that the McLafferty fragmentation occurs within the terbody complex initially formed, with no prior ligand exchange, the water molecule acting as a spectator partner.

Distribution of diarrhetic shellfish poisoning toxins in consignments of blue mussel.

Data describing the distribution of diarrhetic shellfish poisoning toxins in 13 consignments of Danish-produced blue mussels are reported. The content of diarrhetic shellfish poisoning toxins was measured by a liquid chromatography coupled with tandem mass spectrometry detection method, and mean levels in the 13 consignments varied from 58 to 243 microg kg(-1). The distributions of diarrhetic shellfish poisoning toxins in the consignments were relatively homogeneous as the relative standard deviation of the content varied from 7 to 19%. The results are discussed in relation to food safety, the uncertainty of sampling and analysis, and the newly introduced European Union maximum levels of marine biotoxins in seafood products.

Response rates to oestrogen treatment in perimenopausal women: 5-year data from the Danish Osteoporosis Prevention Study (DOPS).

OBJECTIVE: To characterise women with no response or with a good response to hormone replacement therapy (HRT), evaluated by change in bone mineral density (BMD). DESIGN: Nested case-control study within a comprehensive cohort study. SUBJECTS AND METHODS: In the Danish Osteoporosis Prevention Study (DOPS), perimenopausal women were allocated to either HRT or no HRT. In the present study, we included 466 women who had been treated with HRT for 5 years and 466 untreated women from the same cohort. Non-responders were women in the treatment group, who decreased in BMD more than the mean decrease observed in the untreated group. Good responders were women with a larger increase in BMD than the upper 95% percentile of untreated women. Baseline characteristics were evaluated as predictors of response to HRT. RESULTS: 8.4 and 5.6% were classified as non-responders, whereas 25 and 57% were good responders according to changes in BMD of the femoral neck and lumbar spine, respectively. Combining measuring sites, 2.6% were non-responders and 20% were good responders. Non-responders at the femoral neck were more often smokers and had a lower spine BMD. Good responders were older, had a higher body weight, and higher alcohol consumption. In addition, good responders at both measurements sites had a lower BMD at the total hip. CONCLUSION: A favourable BMD response to HRT can be expected in most post-menopausal women especially if they are non-smokers with a moderate--as opposed to low--alcohol intake, a high body mass and a low initial hip BMD.

Long term prognosis of fatty liver: risk of chronic liver disease and death.

BACKGROUND AND AIMS: Fatty liver is a common histological finding in human liver biopsy specimens. It affects 10-24% of the general population and is believed to be a marker of risk of later chronic liver disease. The present study examined the risk of development of cirrhotic liver disease and the risk of death in a cohort diagnosed with pure fatty liver without inflammation. METHODS: A total of 215 patients who had a liver biopsy performed during the period 1976-1987 were included in the study. The population consisted of 109 non-alcoholic and 106 alcoholic fatty liver patients. Median follow up time was 16.7 (0.2-21.9) years in the non-alcoholic and 9.2 (0.6-23.1) years in the alcoholic group. Systematic data collection was carried out by review of all medical records. All members of the study cohort were linked through their unique personal identification number to the National Registry of Patients and the nationwide Registry of Causes of Death, and all admissions, discharge diagnoses, and causes of death were obtained. RESULTS: In the non-alcoholic fatty liver group, one patient developed cirrhosis during the follow up period compared with 22 patients in the alcoholic group. Survival estimates were significantly (p<0.01) different between the two groups, for men as well as for women, with a higher death rate in the alcoholic fatty liver group. Survival estimates in the non-alcoholic fatty liver group were not different from the Danish population. CONCLUSIONS: This study revealed that patients with type 1 non-alcoholic fatty liver disease have a benign clinical course without excess mortality.