Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Occurrence and diversity of ciliated protozoa in the most commonly consumed leafy greens in Brazil: a qualitative study / Ocorrência e diversidade de protozoários ciliados nos vegetais folhosos mais consumidos no Brasil: um estudo qualitativo

Ciliated protozoa are an ubiquitous group of eukaryotes that have been poorly reported on leafy greens. The present study aimed to verify the occurrence and diversity of ciliated protozoa in the leaves and roots of three of the most commonly consumed leafy greens in Brazil – lettuce, rocket and coriander. The vegetable samples were washed by manual agitation (3 minutes) in two different media (mineral water and Page ́s Amoeba Saline solution). After washing, the contents were incubated in Petri dishes and aliquots were removed for microscopic identification and in vivo observation. A total of 21 ciliated protozoa species were found, most of which were bacterivorous. Leafy greens have commonly been associated with foodborne outbreaks and ciliated protozoa, which although they are not a Public Health concern, can act as “Trojan Horses” harboring bacteria, viruses and other protozoa cysts and oocysts and can suggest a new route towards microbiological quality related to the food chain. This is the first report of ciliated protozoa on leafy greens consumed in Brazil.

Os protozoários ciliados constituem um grupo onipresente de eucariotos pouco relatados em vegetais folhosos. O presente estudo teve como objetivo verificar a ocorrência e a diversidade de protozoários ciliados nas folhas e raízes de três dos vegetais folhosos mais consumidos no Brasil - alface, rúcula e coentro. As amostras de vegetais foram lavadas por agitação manual (3 minutos) em dois meios diferentes (água mineral e Solução Salina para Ameba). Após a lavagem, o conteúdo foi incubado em placas de Petri e alíquotas foram retiradas para identificação microscópica e observação in vivo dos organismos. Um total de 21 espécies de protozoários ciliados foi encontrado, a maioria das quais era bacterívora. Tais hortaliças têm sido comumente associadas a surtos transmitidos por alimentos e os protozoários ciliados que, embora não sejam um problema de saúde pública, podem atuar como “Cavalos de Tróia” ao abrigar bactérias, vírus além de cistos e oocistos de protozoários o que pode sugerir uma nova rota para a avaliação da qualidade microbiológica relacionada à cadeia alimentar. Este é o primeiro relato de protozoários ciliados em folhas verdes consumidas no Brasil.

Arbuscular mycorrhizal fungi are an alternative to the application of chemical fertilizer in the production of the medicinal and aromatic plant Coriandrum sativum L.

The widespread use of agrochemicals is detrimental to the environment and may exert harmful effects on human health. The consumer demand for organic food plants has been increasing. There is thus a rising need for alternatives to agrochemicals that can foster sustainable plant production. The aim of this study was to evaluate the potential use of an arbuscular mycorrhizal (AM) fungus as an alternative to application of chemical fertilizer for improving growth performance of the medicinal and aromatic plant Coriandrum sativum. Plants were inoculated with the AM fungus Rhizophagus irregularis BEG163 and/or supplemented with a commercial chemical fertilizer (Plant Marvel, Nutriculture Bent Special) in agricultural soil. Plant growth, nutrition, and development of AM fungus were assessed. Plants inoculated with R. irregularis and those supplemented with chemical fertilizer displayed significantly improved growth performances when compared with controls. There were no significant differences in total fresh weight between plants inoculated with R. irregularis or those supplemented with chemical fertilizer. Leaf chlorophyll a + b (82%), shoot nitrogen (44%), phosphorus (254%), and potassium (27%) concentrations increased in plants inoculated with R. irregularis compared to controls. Application of chemical fertilizer inhibited root mycorrhizal colonization and the length of the extraradical mycelium of R. irregularis. Inoculation with R. irregularis was equally or more efficient than application of chemical fertilizer in promoting growth and nutrition of C. sativum. AM fungi may thus contribute to improve biologically based production of food plants and reduce the dependence on agrochemicals in agriculture.

Decontamination of Salmonella, Shigella, and Escherichia coli O157:H7 from leafy green vegetables using edible plant extracts.

UNLABELLED: Fresh cilantro, parsley, and spinach are products that are regularly consumed fresh, but are difficult to decontaminate, as a result, they are common vehicles of transmission of enteropathogenic bacteria. In this study, the efficacy of plant extracts as alternatives for disinfection of cilantro, parsley, and spinach that were artificially contaminated with Salmonella, Escherichia coli O157:H7, and Shigella sonnei was determined. Edible plant extracts obtained using ethanol as the extraction solvent were tested to determine the minimum bactericidal concentration (MBC) and those that exhibited the lowest MBC were selected for further studies. Leaves of fresh greens were washed with sterile water and dried. For seeding, leaves were submerged in suspensions of 2 different concentrations of bacteria (1.5 × 10(8) and 1 × 10(5) ), dried, and then stored at 4 °C until use. To determine the effects of the extracts, inoculated leafy greens were submerged in a container and subjected to treatments with chlorine, Citrol®, or selected plant extracts. Each treatment type was stored at 4 °C for 0, 1, 5, and 7 d, and the bacterial counts were determined. From the 41 plant extracts tested, the extracts from oregano leaves and from the peel and pulp of limes were found to be as effective as chlorine or Citrol® in reducing by > 2 logs, the population of pathogenic bacteria on leafy greens and therefore, may be a natural and edible alternative to chemicals to reduce the risk of Salmonella, E. coli O157:H7 and S. sonnei contamination on leafy vegetables. PRACTICAL APPLICATION: The antimicrobial efficacy of the extracts of Mexican lime and oregano was clearly demonstrated on cilantro, parsley, and spinach. The extracts of Mexican lime and oregano provide alternatives to chlorine to significantly reduce bacterial pathogens that have been associated with outbreaks from contaminated leafy green vegetables. A simple, low cost, and labor-saving extraction system for production of the extracts was used.

Detection and isolation of low levels of E. coli O157:H7 in cilantro by real-time PCR, immunomagnetic separation, and cultural methods with and without an acid treatment.

UNLABELLED: Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. PRACTICAL APPLICATION: Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.

Fates of seeded Escherichia coli O157:H7 and Salmonella on selected fresh culinary herbs during refrigerated storage.

The fates of seeded Escherichia coli O157:H7 and Salmonella on selected fresh culinary herbs were evaluated at a refrigerated temperature (4 degrees C). Fresh herbs, including cilantro, oregano, basil, chive, parsley, and rosemary, were inoculated with six-strain mixtures of E. coli O157:H7 and Salmonella, and the microbial populations were monitored at 1, 5, 11, 16, 19, and 24 days. For both pathogens, a significant decrease in the population (P < 0.0001) occurred within the first 5 days of storage (< 0.8 log). Both pathogens remained the highest on cilantro and the lowest on rosemary (P < 0.0001). Storage time had a significant effect on the survival of E. coli O157:H7; populations declined as storage time progressed. Although storage of cilantro, basil, and chive was terminated after 19 days because of deteriorated quality, significant numbers of both pathogens were recovered from the remaining fresh herbs after 24 days of storage. The results showed that both bacteria were extremely persistent on all test herbs under the test conditions. The results also reinforce the concept that, once contaminated, bacterial pathogens can persist on fresh herbs throughout a normal distribution time.