Session Type: 1,5-hour Oral Session
Session Title: 1,5-hour Oral Session
Authors(s): C. Moguet (1), S. Gelhaye (1), A. Sallustrau (2), C. Gonzalez (3), T. Naas (4), S. Simon (1), H. Volland (1)
Authors Affiliations(s): (1) Universite Paris-Saclay, CEA, INRAE, DMTS, SPI, France, (2) Universite Paris-Saclay, CEA, INRAE, DMTS, SCBM, France, (3) Bacteriology-Hygiene unit, AP-HP, Hôpital Bicêtre, France, (4) Bacteriology-Hygiene unit, AP-HP, Hôpital Bicêtre & Team Resist, UMR1184, Universite Paris-Saclay - INSERM - CEA, LabEx Lermit, France
Background:
The increasing rates of infections caused by β-lactamases-producing Enterobacteriaceae is a major public health concern. The immunochromatographic assay (ICA) for the detection of β-lactamases from bacterial isolates is already used. Here, we have developed and validated a multiplex ICA allowing the detections of the expanded-spectrum cephalosporinase (ESCase) activity and the five groups of CTX-Ms.
Methods:We selected and produced monoclonal antibodies (mAbs) specific for the intact cefotaxime molecule and to the five groups of CTX-Ms. On the ICA, a first test line (TL1) that detects an ESCase activity, a second test line that detects CTX-M enzymes and a control line capturing all excess mAbs with goat anti-mouse mAbs (Figure 1). One single colony was resuspended in 150 µL of extraction buffer containing 25ng/mL of cefotaxime. After 30-minute incubation at RT, 100 µL was loaded on the ICA and the results were eye read after 10-minutes migration. In absence of ESCase, the cefotaxime binds the anti-cefotaxime mAb’s paratopes labelled with colloidal gold. These mAbs cannot bind to the cefotaxime immobilized on the TL1 : no signal is detected. In presence of ESCase activity, the hydrolyzed cefotaxime is not recognized by the anti-cefotaxime mAbs, which bind to the immobilized cefotaxime : positive signal on TL1. The detection of CTX-Ms is made using an ICA previously developed1. This multiplex ICA was retrospectively evaluated on 161 bacterial isolates from the F-NRC.
Results:Irrespective of the bacterial species or the ß-lactamase gene, all isolates known to display an ESCase activity and/or to produce a CTX-M enzyme were detected. As a result, the sensitivity and specificity obtained were 100% for ESCase activity- and CTX-M- detection.
Conclusions:This test allows a reliable and fast detection of CTX-M enzymes and ESCase activity in less than 40 minutes. By combining these two detection methods, we are able to simultaneously detect the presence of an ESCase activity and whether this activity is related to the presence of a CTX-M enzyme (represent 98% of ESBLs currently spreading in France). This test is low cost and easy to implement in routine laboratory workflow as first-line test on bacterial colonies.
Keyword(s): Lateral flow immunoassay, Béta-lactamase detection, Cephalosporin hydrolysisCOI Other: A patent has been filed (B252171FR D41054) to protect this invention, in the name of three inventors: Dr. Hervé Volland, Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France; Mr. Christian Moguet, Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France; Dr. Thierry Naas, Team Resist, UMR1184, Université Paris-Saclay - INSERM - CEA, LabEx Lermit, Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-producing Enterobacterales, Le Kremlin-Bicêtre, France.
Session Type: 1,5-hour Oral Session
Session Title: 1,5-hour Oral Session
Authors(s): C. Moguet (1), S. Gelhaye (1), A. Sallustrau (2), C. Gonzalez (3), T. Naas (4), S. Simon (1), H. Volland (1)
Authors Affiliations(s): (1) Universite Paris-Saclay, CEA, INRAE, DMTS, SPI, France, (2) Universite Paris-Saclay, CEA, INRAE, DMTS, SCBM, France, (3) Bacteriology-Hygiene unit, AP-HP, Hôpital Bicêtre, France, (4) Bacteriology-Hygiene unit, AP-HP, Hôpital Bicêtre & Team Resist, UMR1184, Universite Paris-Saclay - INSERM - CEA, LabEx Lermit, France
Background:
The increasing rates of infections caused by β-lactamases-producing Enterobacteriaceae is a major public health concern. The immunochromatographic assay (ICA) for the detection of β-lactamases from bacterial isolates is already used. Here, we have developed and validated a multiplex ICA allowing the detections of the expanded-spectrum cephalosporinase (ESCase) activity and the five groups of CTX-Ms.
Methods:We selected and produced monoclonal antibodies (mAbs) specific for the intact cefotaxime molecule and to the five groups of CTX-Ms. On the ICA, a first test line (TL1) that detects an ESCase activity, a second test line that detects CTX-M enzymes and a control line capturing all excess mAbs with goat anti-mouse mAbs (Figure 1). One single colony was resuspended in 150 µL of extraction buffer containing 25ng/mL of cefotaxime. After 30-minute incubation at RT, 100 µL was loaded on the ICA and the results were eye read after 10-minutes migration. In absence of ESCase, the cefotaxime binds the anti-cefotaxime mAb’s paratopes labelled with colloidal gold. These mAbs cannot bind to the cefotaxime immobilized on the TL1 : no signal is detected. In presence of ESCase activity, the hydrolyzed cefotaxime is not recognized by the anti-cefotaxime mAbs, which bind to the immobilized cefotaxime : positive signal on TL1. The detection of CTX-Ms is made using an ICA previously developed1. This multiplex ICA was retrospectively evaluated on 161 bacterial isolates from the F-NRC.
Results:Irrespective of the bacterial species or the ß-lactamase gene, all isolates known to display an ESCase activity and/or to produce a CTX-M enzyme were detected. As a result, the sensitivity and specificity obtained were 100% for ESCase activity- and CTX-M- detection.
Conclusions:This test allows a reliable and fast detection of CTX-M enzymes and ESCase activity in less than 40 minutes. By combining these two detection methods, we are able to simultaneously detect the presence of an ESCase activity and whether this activity is related to the presence of a CTX-M enzyme (represent 98% of ESBLs currently spreading in France). This test is low cost and easy to implement in routine laboratory workflow as first-line test on bacterial colonies.
Keyword(s): Lateral flow immunoassay, Béta-lactamase detection, Cephalosporin hydrolysisCOI Other: A patent has been filed (B252171FR D41054) to protect this invention, in the name of three inventors: Dr. Hervé Volland, Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France; Mr. Christian Moguet, Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France; Dr. Thierry Naas, Team Resist, UMR1184, Université Paris-Saclay - INSERM - CEA, LabEx Lermit, Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-producing Enterobacterales, Le Kremlin-Bicêtre, France.