The increasing consumption of organic or ready-to-eat food may cause serious foodborne disease outbreaks. Developing microbiological culture for detection of food-borne pathogens is time-consuming, expensive, and laborious. Thus, alternative methods such as polymerase chain reaction (PCR) are usually employed for outbreaks investigation. In this work, we aimed to develop a rapid and simple protocol for the simultaneous detection of Escherichia coli (E coli), Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus) and Salmonella enterica (S. enterica), by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered peptone water (BPW) was selected as the optimum one. Then, mPCR conditions were optimized and applied both to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In the culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR assy was able to detect E. coli, S. enterica, and L. monocytogenes. In conclusion, BPW broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes, and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, thereby considerably reducing the time, efforts and costs of analyzes.
The current strategy for the control of helminth infections relies on chemotherapy. However, resistance appearance is promoting the necessity of developing new drugs against trematodes. Herein, potential trematocidal effects of garlic (Allium sativum) are investigated in the context of intestinal foodborne trematodes, employing the Echinostoma caproni-mouse model. Daily administration of dietary doses of garlic was conducted in three groups of mice: (i) before infection (prophylaxis), (ii) after infection (therapeutic) and (iii) both, before and after infection (continuous). A fourth group of mice, not exposed to garlic, was used as control. No differences in worm recovery, fecundity and local cytokine expression profiles were found with respect to control infections. However, considerable alterations in tegument structure, including swelling, furrowing, vacuolization and changes in secretory bodies were detected in garlic-exposed parasites using scanning and transmission electron microscopy. Protein secretion was markedly reduced in response to garlic, whereas up-regulation of several proteins, such as major vault protein and tER-ATPase, was observed in treated worms. The results presented herein provide new insights in the anthelminthic activity of bioactive garlic compounds and the manner that parasites respond to toxins.
Vegetables are one of the main foodstuffs consumed in the Mediterranean diet. However, raw vegetables have been associated with relevant foodborne outbreaks worldwide. Accurate knowledge of the microbiological quantitative risks associated with these matrices is crucial in order to define effective control measures, avoiding the survival and dissemination of foodborne pathogens through the different food chain stages. The aim of the present study is the assessment of the prevalence of Helicobacter pylori (a unique carcinogenic biological agent recognized to date) on leafy vegetables (spinach, lettuce, and chard) by means of the detection of the specific pathogenicity vacA gene. A real-time quantitative polymerase chain reaction (qPCR) optimized approach was used to detect H. pylori-positive samples and the concentration of this pathogen (with a limit of detection equal to 10 cells). One hundred raw vegetable samples were acquired in markets corresponding to the Spanish Mediterranean area. Sliced vegetable leaves were homogenized and centrifuged, and DNA was extracted from the homogenates. qPCR results confirmed 20 out of 100 H. pylori-positive samples, with melting temperature (Tm) values in the range of 84.8–86.5 °C (Tm vacA H. pylori = 85 °C). Amplicons were cut, purified, and sequenced to confirm the homology with the H. pylori vacA gene. A total of 17 out of 100 vegetable samples (12/45 (26.6%) lettuce, 2/21 (9.5%) spinach, and 3/34 (8.8%) chard samples) were finally confirmed as H. pylori-positive. Contamination levels were in the range of 1.5 ± 0.3 to 2.5 ± 0.1 log10 cycles (36–335 CFU/g leafy vegetables). Our results show that H. pylori is detected by qPCR at levels close to infectious doses in fresh vegetables, thus posing a food safety hazard.
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