Amalia Martinez-Perez

Development of a method for the detection of verotoxin-producing Escherichia coli in food.

The growing recognition of the role of non-O157 verotoxigenic Escherichia coli (VTEC) in foodborne illness underscores the importance of developing methods to detect it in the food supply. We describe here the development of a protocol for the detection, isolation, and characterization of VTEC from foods, designed for the serotype-independent enrichment, detection, and isolation of VTEC, in combination with rapid characterization of VTEC O157, O26, O103, O111, and O145. This study examined the inhibitory concentration of six antimicrobial agents used either singly or in combination for the optimal enrichment of a panel of 18 different O serogroups of VTEC in modified tryptic soy broth. Considerable variability in resistance to the different antimicrobials tested was noted among different VTEC strains. The combination enabling growth of strains of all 18 different O serogroups was vancomycin (10 μg/ml) and cefsulodin (3 μg/ml). A similar combination of antimicrobials formulated in agar plates was found beneficial in the recovery of VTEC strains from enrichment broth cultures. The efficacy of these media in the recovery of selected VTEC (O26, O103, O111, O145, and O157) from ground beef and O157 VTEC from lettuce, spinach, and apple cider was demonstrated. The selective enrichment media described herein would appear suitable for incorporation in methods for the recovery and detection of a wide range of VTEC serogroups.

Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin‐based ELISA

Aims:  Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin‐based enzyme‐linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens.

A simple PCR-based macroarray system for detection of multiple gene markers in the identification of priority enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains bearing the O antigenic determinants O157, O26, O111, O103, and O145 have a high rate of association with foodborne illness worldwide. To expand Canadian food inspection capability, a cloth-based hybridization array system (CHAS) was developed for the identification and characterization of priority EHEC. This method targets key virulence genes (eae, hlyA, vt1, and vt2) plus the rfbE gene specifying the O157 antigenic determinant, and the wzx genes specifying the O26, O111, O103, and O145 determinants. Multiplex PCR products incorporating a digoxigenin label were detected by hybridization with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. This method identified the relevant markers in 85 different strains bearing various combinations of the target genes (virulence and priority O-antigen markers). None of the target genes was detected in 26 different strains of other E. coli and non-E. coli bacteria. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the various markers among different bacterial strains. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the markers among various target and nontarget bacteria. The entire procedure could be completed in less than 5 h, and is useful for the identification of priority EHEC colonies isolated from foods by using enrichment culture techniques.

Detection of group D salmonellae including Salmonella Enteritidis in eggs by polymyxin-based enzyme-linked immunosorbent assay.

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9-specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

Development of Unique Bacterial Strains for Use as Positive Controls in the Food Microbiology Testing Laboratory

Nalidixic acid-resistant (NalR) mutants of Salmonella enterica serovar Berta and Escherichia coli O157:H7 were derived from wild-type laboratory cultures to serve as distinguishable control strains for routine use in food microbiology testing programs. The prevalence of the NalR phenotype among different bacteria was verified using panels of related and unrelated strains with the ability to grow vigorously on plating media containing nalidixic acid, being restricted to the NalR mutants. The NalR phenotype was stable in both mutant strains over several generations in the absence of selective pressure and enabled their differentiation from wild-type bacteria on the basis of their ability to grow on plating media containing nalidixic acid. A similar approach for the development of a distinguishable Listeria monocytogenes control strain was not possible due to the inherent resistance of this organism to nalidixic acid. Instead, an L. monocytogenes isolate with rare genotypic and serologic features was identified as a possible candidate to serve as a unique and distinguishable positive control strain.

Risk Profile on Non-O157 Verotoxin- Producing Escherichia Coli in Produce, Beef, Milk and Dairy Products in Canada

This risk profile on non-O157 verotoxin-producing Escherichia coli serotypes (non-O157 VTEC) in produce, beef, milk and dairy products in Canada, outlines associated potential public health threats, while identifying areas where additional data are required to inform microbiological risk assessment and risk management action. The following outputs of this risk profile may contribute to risk analysis relating to non-O157 VTEC in Canada: • Beef products, milk and dairy products, and produce are all food commodities associated with non-O157 illness outbreaks. Some non-O157 VTEC may cause foodborne illnesses similar to E. coli O157:H7, however in general, non-O157 VTEC cause less severe illness or lead to asymptomatic infections. In the absence of epidemiological evidence (i.e., the inability to show that the detected bacteria has previously caused illness in humans), the ability to establish the health concern of isolates, is limited. • Non-O157 VTEC serogroups most commonly implicated in human infections differ between countries. In Canada, the top six non-O157 VTEC serogroups associated with serious illness are: O26, O103, O111, O117, O121, and O145. • The dose-response(s) for non-O157 VTEC in beef products, milk and dairy, and produce as groups is currently not possible to determine. • VTEC are defined by the production of verotoxins. Other virulence factors are associated with clinical isolates of non-O157 VTEC and may indicate an increased threat to human health, but a common virulence profile for pathogenic VTEC has not been identified. • The government of Canada commissioned the Federal VTEC Working Group to develop detection and isolation methods for non-O157 VTEC as an initial regulatory response to support the reporting of non-O157 VTEC in foods. A Canadian VTEC method collaboratively developed by Health Canada and the Canadian Food Inspection Agency is being used to detect the presence of non-O157 VTEC in food. 1 Angela Catford, Valentin Kouame, Amalia Martinez-Perez, Alexander Gill, Enrico Buenaventura, Helene Couture and Jeffrey M Farber: Risk Profile on Non-O157 Verotoxin-Producing Escherichia Coli in Produce, Beef, Milk and Dairy Products in Canada ARTICLE Int Food Risk Anal J, 2014, 4:21 | doi: 10.5772/59208 International Food Risk Analysis Journal