Yersinia enterocolitica detection in pork products: Evaluation of isolation protocols.

Conventional methods for Yersinia enterocolitica detection in food samples are generally considered inadequate. Problems arise from the presence of the so-called "background flora", coupled to the low contamination level of the pathogen. Since, data on the microbial ecology occurring in competitive microflora are still lacking, MALDI TOF MS was used for strains 'identification after enrichment in PSB or ITC broths, and after plating on selective CIN medium at different incubation times. SYBR Green Real time PCR was used for the Y. enterocolitica strains' detection (4/O:3, 1A/O:5) in experimentally contaminated foods, as well as in naturally contaminated samples. A higher number of different bacterial genera (10 on CIN and 18 on PCA) was recorded after enrichment in PSB, whilst enrichment in ITC led to recovery of 6 and 10 genera on CIN and PCA, respectively. Yersiniaceae was the dominant family on the first day of incubation, but on the second day the percentage of isolation considerably decreased. By testing experimentally contaminated samples, substantial difficulties were encountered. The biotype 1A was always detected, whereas strain 4/O:3 proved to be poorly competitive. Based on the data, the enrichment media PSB and ITC, currently proposed for Y. enterocolitica detection, need to be improved to promote a successful pathogen's recovery.

ELIME assay vs Real-Time PCR and conventional culture method for an effective detection of Salmonella in fresh leafy green vegetables.

The detection of Salmonella according to EC regulation is still primarily based on traditional microbiological culture methods that may take several days to be completed. The purpose of this work is to demonstrate the applicability of an Enzyme-Linked-Immuno-Magnetic-Electrochemical (ELIME) assay, recently developed by our research group for the detection of salmonella in irrigation water, in fresh (raw and ready-to-eat) leafy green vegetables by comparison with Real-Time PCR (RTi-PCR) and ISO culture methods. Since vegetables represent a more complex matrix than irrigation water, preliminary experiments were carried out on two leafy green vegetables that resulted negative for salmonella by the ISO method. 25g of these samples were experimentally inoculated with 1-10 CFU of S. Napoli or S. Thompson and pre-enriched for 20h in two different broths. At this time aliquots were taken, concentrated at different levels by centrifugation, and analyzed by ELIME and RTi-PCR. Once selected the best culture medium for salmonella growth, and the optimal concentration factor suitable to reduce the sample matrix effect, enhancing the out-put signal, several raw and ready-to-eat leafy green vegetables were artificially inoculated and pre-enriched. Aliquots were then taken at different incubation times and analyzed with both techniques. Results obtained showed that 20 and 8h of pre-enrichment were required to allow the target salmonella (1-10 CFU/25g) to multiply until reaching a detectable concentration by ELIME and RTi-PCR assays, respectively. A confirmation with the ISO culture method was carried out. Based on the available literature, this is the first report of the application of an ELISA based method for the detection of Salmonella in vegetables.

Development and evaluation of an ELIME assay to reveal the presence of Salmonella in irrigation water: Comparison with Real-Time PCR and the Standard Culture Method.

A reliable, low-cost and easy-to-use ELIME (Enzyme-Linked-Immuno-Magnetic-Electrochemical) assay for detection of Salmonella enterica in irrigation water is presented. Magnetic beads (MBs), coupled to a strip of eight-magnetized screen-printed electrodes localized at the bottom of eight wells (8-well/SPE strip), effectively supported a sandwich immunological chain. Enzymatic by-product is quickly measured by chronoamperometry, using a portable instrument. With the goal of developing a method able to detect a wide range of Salmonella serotypes, including S. Napoli and S. Thompson strains responsible for various community alerts, different kinds of MBs, antibodies and blocking agents were tested. The final system employs MBs coated with a broad reactivity monoclonal antibody anti-salmonella and blocked with dry milk. For a simple and rapid assay these two steps were performed in a preliminary phase, while the two sequential incubations for the immuno-recognition events were merged in a single step of 1h. In parallel a Real-Time PCR (RTi-PCR) method, based on a specific locked nucleic acid (LNA) fluorescent probe and an internal amplification control (IAC), was carried out. The selectivity of the ELIME and RTi-PCR assays was proved by inclusivity and exclusivity tests performed analyzing different Salmonella serotypes and non-target microorganisms, most commonly isolated from environmental sources. Furthermore, both methods were applied to experimentally and not experimentally contaminated irrigation water samples. Results confirmed by the ISO culture method, demonstrated the effectiveness of ELIME and RTi-PCR assays to detect a low number of salmonella cells (1-10 CFU/L) reducing drastically the long analysis time usually required to reveal this pathogen.

Microbiological survey of raw and ready-to-eat leafy green vegetables marketed in Italy.

The presence of foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, thermotolerant Campylobacter, Yersinia enterocolitica and norovirus) in fresh leafy (FL) and ready-to-eat (RTE) vegetable products, sampled at random on the Italian market, was investigated to evaluate the level of risk to consumers. Nine regional laboratories, representing 18 of the 20 regions of Italy and in which 97.7% of the country's population resides, were involved in this study. All laboratories used the same sampling procedures and analytical methods. The vegetable samples were screened using validated real-time PCR (RT-PCR) methods and standardized reference ISO culturing methods. The results show that 3.7% of 1372 fresh leafy vegetable products and 1.8% of 1160 "fresh-cut" or "ready-to-eat" (RTE) vegetable retailed in supermarkets or farm markets, were contaminated with one or more foodborne pathogens harmful to human health.

Towards the development of a single-step immunosensor based on an electrochemical screen-printed electrode strip coupled with immunomagnetic beads.

This work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG-HRP. The assay protocol is based on the use of a series of small "reservoirs" containing phosphate buffer, hydroquinone, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each "reservoir" is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H(2)O(2) produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H(2)O(2), is measured. The second, more direct, approach is performed without exploiting ECP (no GOD enzyme), by adding H(2)O(2) into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimination between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the background signal due to HRP in the bulk solution.

An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler.

Rapid and specific detection of botulinum neurotoxin (BoNT) producing Clostridia is a priority for public health authorities, in case of both natural and intentional botulism outbreaks. This study reports on the evaluation of a detection system based on the GeneDisc Cycler designed for simultaneously testing the bont/A, bont/B, bont/E and bont/F genes encoding for the botulinum neurotoxins types A, B, E and F. BoNT-producing Clostridia (n = 102) and non-BoNT-producing bacteria (n = 52) isolated from clinical, food and environmental samples were tested using this macro-array and results were compared to the reference lethality test on mice. The bont genes were correctly detected in all C. botulinum type A, B, E and F strains available, as well as in toxigenic C. baratii type F and toxigenic C. butyricum type E. No cross reactivity was observed with non human-toxigenic bacteria, C. botulinum types C, D and G. The identification of the bont genotype using the macro-array was correlated to toxino-typing of the BoNTs as determined by the mouse bioassay. An "evaluation trial" of the GeneDisc array performed blind in four European laboratories with 77 BoNT-producing Clostridia as well as 10 food and clinical samples showed that the developed macro-array is specific and reliable for identifying BoNT/A-, BoNT/B-, BoNT/E- and BoNT/F-producing clostridial strains and for screening naturally contaminated food and fecal samples. The test is robust, has a low detection limit (c.a. 5 to 50 genome copies in the PCR reaction microwell) and is promising for monitoring BoNT-producing Clostridia in different kinds of samples including food and clinical samples.