On-chip microbial culture for the specific detection of very low levels of bacteria.

Bouguelia S(1), Roupioz Y, Slimani S, Mondani L, Casabona MG, Durmort C, Vernet T, Calemczuk R, Livache T.

Lab Chip. 2013 Oct 21;13(20):4024-32. doi: 10.1039/c3lc50473e.

Microbial culture continues to be the most common protocol for bacterial detection and identification in medicine and agronomics. Using this process may take days to identify a specific pathogen for most bacterial strains. Surface Plasmon Resonance (SPR) detection is an emerging alternative technology that can be used for the detection of bacteria using protein microarrays although typical limits of detection are in the range of 10(3)-10(6) cfu mL(-1), which is not compatible with most Food Safety regulation requirements. In this work, we combine concomitant “on-chip” microbial culture with sensitive SPR detection of bacteria thus allowing rapid specific detection of bacteria pathogens – including Salmonella enterica serovar Enteritidis, Streptococcus pneumoniae and Escherichia coli O157:H7 – cultured on a protein microarray. This Culture-Capture-Measure (CCM) approach significantly decreases both the number of processing steps and the overall assay time for bacterial detection. Signal analysis of SPR responses allowed the fast and quantitative assessment of bacterial concentrations initially present in the sample as low as 2.8 ± 19.6 cfu per milliliter. Altogether, our results show how simple, easy-to-operate, fluidic-less and lo-tec microarrays can be used with unprocessed samples and yield – in a single assay – both qualitative and quantitative information regarding bacterial contamination.


Immunological detection of Cronobacter and Salmonella in powdered infant formula by plasmonic label-free assay.

Morlay A, Piat F, Mercey T, Roupioz Y.

Lett Appl Microbiol. 2016 Jun;62(6):459-65. doi: 10.1111/lam.12570

Cronobacter is an emerging food pathogen, especially in infants and neonates, often associated with the ingestion of contaminated Powdered Infant Formula (PIF). Therefore, regulations require the control of the absence of Cronobacter and of Salmonella, another important food pathogen, in these food products. So far, reference and alternative methods take up to several days, and no validated method exists for the simultaneous detection of these two pathogens. In this work, we propose to address this issue by an innovative and easy-to-operate assay, named Plasmonic Immuno-Assay (PlasmIA), and by producing dedicated polyclonal antibodies. Our approach is based on Surface Plasmon Resonance imaging of antibody-arrays and bacterial growth during a standardized enrichment. Such a single-step assay enables the multiplex detection of both Cronobacter and Salmonella, with concentrations smaller than 30 CFU cells in 25 g PIF samples, in less than 1 day.

Label-free immuno-sensors for the fast detection of Listeria in food.

Alexandra Morlay, Aurore Duquenoy, Félix Piat, Roberto Calemczuk, Thibaut Mercey, Thierry Livache, Yoann Roupioz.

Measurement, 2016

Listeria monocytogenes is one of the major foodborne pathogen found in a large set of foodstuff. With the aim of decreasing listeriosis occurrence, the early detection of this pathogen in food products is of tremendous interest. So far, the reference method and all the alternative methods require a pre-enrichment step lasting 24 h to several days. Then, an aliquot of the enriched sample is collected and characterized according different methods including genetic analysis, or beads immuno-agglutination. With the aim of significantly decreasing the overall processing time, we propose an approach involving the detection of alive bacteria during their growing phase via the use of an immuno-chip SPR imaging process. Listeria specific antibodies were also produced and chemically grafted on the biochip before being selected for their ability to distinguish different Listeria strains. Finally, this approach allows the detection of a very low number of L. monocytogenes present in foodstuff.

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

Alexandra Morlay, Agnès Roux, Vincent Templier , Félix PIAT, Yoann Roupioz.

Microbial Toxins: Methods and Protocols, Methods Mol Biol., vol.1600

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

Fast detection of both O157 and non-O157 shiga-toxin producing Escherichia coli by real-time optical immunoassay.

Mondani L, Delannoy S, Mathey R, Piat F, Mercey T, Slimani S, Fach P, Livache T, Roupioz Y.

Lett Appl Microbiol. 2016 Jan;62(1):39-46. doi: 10.1111/lam.12503.

Among bacterial pathogens involved in food-illnesses, seven serogroups (O26, O45, O103, O111, O121, O145 and O157) of Shiga-toxin producing Escherichia coli (STEC), are frequently identified. During such outbreak, and due to the perishable property of most foodstuff, the time laps for the identification of contaminated products and pathogens is thus critical to better circumvent their spread. Traditional detection methods using PCR or culture plating are time consuming and may present some limitations. In this study, we present a multiplexed immunoassay for the optical detection of most commonly enterohemorrhagic E. coli serogroups: O26, O45, O103, O111, O121, O145 and O157:H7 in a single device. The use of Surface Plasmon Resonance imaging not only enabled the label-free analysis of the samples but gave results in a real-time manner. A dedicated protocol was set up for the detection of both low contaminating bacterial concentrations of food samples (5 CFU per 25 g) and postenrichment aliquots. By combining one single device for the detection of O157 and non-O157 STEC in a label-free manner, this rapid approach may have an important economic and societal impact.


We have presented several posters in international conferences :

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