Helicobacter pylori Is Present at Quantifiable Levels in Raw Vegetables in the Mediterranean Area of Spain

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 (TmvacA 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.