Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation.

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-µm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.

Inhibition of polymerase chain reaction for the detection of Escherichia coli O157:H7 and Salmonella enterica on walnut kernels.

The aim of this study was to determine whether Escherichia coli O157:H7 can be reliably detected and isolated from walnut kernels using standard methods of analysis. The limit of detection approached 1 cell per analytical unit (25 g) for E. coli O157:H7 on walnut kernels enriched in modified tryptic soy broth with 20 µg/ml novobiocin and plating onto selective agar media. The presence of PCR inhibitors in walnut kernels was indicated by the failure to detect E. coli O157:H7 from culture positive enrichment broths analysed by PCR, with two separate polymerase and reagent compositions (Dupont BAX E. coli O157:H7 MP system, Promega GoTaq Green for stx) and three methods of template preparation (DuPont BAX, Qiagen DNeasy, Bio-Rad InstaGene). PCR inhibition was overcome by 1:100 dilution in TE buffer of the DNeasy or InstaGene template. PCR inhibition was not relieved by dilution of the BAX template. Similar results were observed for walnut kernels inoculated with Salmonella enterica and analysed for invA, indicating that PCR inhibition is not specific to the organism or primer/template. These results indicate that analysis of walnut kernels for pathogens should be with culture based methods or use protocols for DNA template preparation modified to remove or dilute inhibitors and the need for internal amplification controls in PCR methods.

Microbial safety of cheese in Canada.

A profile of the microbial safety of cheese in Canada was established based on the analysis of 2955 pasteurized and raw-milk cheeses tested under Canada's National Microbiological Monitoring Program (NMMP) and 2009 raw-milk cheeses tested under the Targeted Survey Program. 97.8% of NMMP and 99.6% of Targeted Survey cheese samples were assessed as being of satisfactory microbiological safety. Under the NMMP, Salmonella spp. was detected in 2 samples, Listeria monocytogenes was detected in 15 samples and no Escherichia coli O157/H7:NM (non-motile) was detected. Cheese samples assessed as having unsatisfactory levels of S. aureus and generic E. coli were found in 18 and 41 samples, respectively. Under the Targeted Survey, L. monocytogenes was detected in 2 samples, while no Salmonella spp. or E. coli O157/H7:NM were detected. Cheese samples assessed as having investigative and unsatisfactory levels of S. aureus were found in 4 and 2 samples respectively. No samples were found to have investigative or unsatisfactory levels of generic E. coli. For cheese samples collected under the NMMP, logistic regression models indicated that contamination was more frequent in raw-milk cheeses compared to pasteurized-milk cheeses (OR = 5.0, 95% CI (3.0, 8.3)), and in imported cheeses compared to domestic cheeses (OR = 8.2, 95% CI (4.1, 16.1)). A statistically significant association was found between cheese samples assessed as having unsatisfactory levels of generic E. coli and detection of L. monocytogenes, Salmonella spp. or levels of S. aureus that were assessed as unsatisfactory (p < .001). These test results will help support risk analysis and inform food safety decisions.

Enumerative Analysis of Salmonella in Outbreak-Associated Breaded and Frozen Comminuted Raw Chicken Products.

A significant data gap exists with respect to the levels of pathogens in foods implicated in foodborne outbreaks. These data are essential for the quantification of pathogen exposure via the ingestion of contaminated food. Here we report the levels of the foodborne pathogen Salmonella in comminuted raw chicken products that had been breaded and then frozen. The products investigated were collected during four food safety investigations of foodborne outbreaks that occurred in Canada from 2014 to 2016. Most-probable-number (MPN) distribution analysis of the food samples revealed Salmonella levels of 0.0018 to 3 MPN/g, which is equivalent to 1 MPN per 0.33 to 556 g of product. These data suggest low levels of Salmonella may be associated with foodborne outbreaks.