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In this study, a total of 175 samples of ready-to-eat vegetables, frozen vegetables and sprouted seeds originating in 10 states of the European Union and from 32 manufacturers were collected during a period of one year and examined for the presence of Listeria monocytogenes using standard culture methods and qPCR. In addition to these methods, isolation of Listeria monocytogenes was also carried out following a unified sample preparation for combined downstream use in culture and qPCR analysis. Standard culture and culture preceded by unified sample preparation, showed that L. monocytogenes was present in 6.9% and 11.4% of analyzed samples, respectively, in low numbers. Application of qPCR revealed only 2.3% of samples to be positive for L. monocytogenes in small quantities (less than 10 cells/gram). A statistically significant higher occurrence of L. monocytogenes was seen in frozen vegetables compared to ready to eat vegetables (p<0.01; Fisher's exact post-hoc tests with Bonferroni's correction) or sprouts (p<0.05; Fisher's exact post-hoc tests with Bonferroni's correction). Therefore, temperature abuse in food containing pieces of frozen vegetables without any processing such as cooking, may pose health risks, especially for sensitive individuals such as pregnant women, children, the elderly, and immunocompromised individuals.

Environmental matrices and food products are hypothesized to be sources of Cronobacter spp. The severity of neonatal infections, increasing number of cases in elderly and immunocompromised individuals, as well as isolation of Cronobacter spp. from clinical materials demands that more attention should be paid to Cronobacter spp. detection and occurrence of the bacteria in food products. Here, a total of 175 samples of ready-to-eat vegetables, frozen vegetables, and sprouted seeds were collected during a period of 1 year and examined for the presence of Cronobacter spp. using a cultivation method with two different sample preparations and real-time polymerase chain reaction (qPCR). In total, Cronobacter spp. were detected in 22.3% of tested samples using cultivation. In comparison, direct qPCR detected Cronobacter spp. in 37.7% of these samples (p < 0.01; Fisher's exact test) and the numbers of genome equivalents per gram reached 10 8 in some samples of sprouts. Cronobacter spp. were isolated from 51.4%, 37.2%, and 5.2% samples of sprouts, frozen vegetables, and cut green leaves/salads, respectively. Using qPCR, the most frequently contaminated sample types were sprouts (91.4%) and frozen vegetables (60.5%), whereas the rate of positivity for cut green leaves/salads was, in comparison, only 8.2% (p < 0.01; χ 2 -test for independence).Practical Application: This study provided valuable information on the occurrence of Cronobacter spp. in ready-to-eat vegetables using cultivation and qPCR. Cronobacter spp. are emerging opportunistic pathogens that can be present in food of plant origin. Cronobacter spp. were isolated from sprouts, frozen vegetables, and cut green leaves/salads, and the numbers of genome equivalents per gram reached 10 8 in some samples of sprouts.

Fresh vegetables and herbs are usually prepared and eaten raw without cooking or heating, which leads to a high risk of foodborne infection. The aim of the present study was to assess the contamination of raw vegetables, herbs, and the environment of food chains. Vegetable and herb samples originating both from the Czech Republic as well as from other countries were examined. The work was focused on the detection of commonly found, but also less frequently monitored foodborne pathogens, including viruses of the genus Norovirus (NoVs), hepatitis A virus (HAV), Listeria monocytogenes and Cronobacter spp. bacteria, and the parasites Cryptosporidium spp. and Giardia intestinalis. All samples were analyzed using individual RT-qPCR/qPCR assays; bacterial pathogens were also simultaneously detected using culture methods. The prevalence of the studied microorganisms in 623 samples ranged from 0.6% to 44.3% for individual pathogens. None of the samples were positive for the presence of HAV. Analysis of 157 environmental samples from 12 farms revealed the presence of NoVs in the environment of four farms. NoVs were detected in water samples as well as on the hands and gloves of workers. Escherichia coli was detected in all farms in the environmental samples and in eight farms in water samples. However, no sample of water exceeded the level of 100 CFU/mL for E. coli. None of the samples of water were positive for the presence of the studied parasites. Vegetables and herbs available from Czech markets and farms may pose a certain risk of foodborne disease, especially in the case of NoVs and parasites.Practical Application: This study provides valuable information on the microbiological quality of raw vegetables and herbs available from Czech markets and farms. Good hygienic practices aimed at reducing the incidence of pathogenic agents on fresh produce should not be neglected. Emphasis should be placed on the control of irrigation water, especially with respect to norovirus contamination. It is appropriate to combine culture methods and qPCR methods for the detection of bacterial agents. Hepatitis A virus; LM, Listeria monocytogenes; Crono. spp., Cronobacter spp.; Crypto. spp., Cryptosporidium spp.; G. int., Giardia intestinalis; EC, Escherichia coli; * , positive samples; * * , median of genomic equivalents in one gram of sample 3046