Method for detecting the presence of expanded spectrum b-lactamase-producing bacteria in a sample

Abstract

The present invention relates to a method for detecting the presence of expanded-spectrum β-lactamase (β-lactamase hydrolyzing expanded-spectrum cephalosporin)-producing bacteria in a sample, said method comprising the steps of: a) performing cell lysis on a test sample in order to obtain an enzymatic suspension; b) reacting a fraction of the enzymatic suspension obtained in step a) with a reagent kit, said reagent kit comprising—expanded-spectrum β-lactamase substrate selected from the group consisting of cephalosporins, aztreonam, and cephamycins, —a pH color indicator which will change color when the pH of the reaction mixture is comprised between 6.4 and 8.4, wherein a color change after step b) indicates the presence of expanded-spectrum β-lactamase-producing bacteria in the test sample. The invention also relates to a reagent kit, to a microtiter plate and to their uses in detecting the presence of expanded-spectrum β-lactamase producers in a test sample.

1 . A kit comprising:
 a lysis buffer; and   a reagent kit comprising
 an expanded-spectrum β-lactamase substrate (ESBL) selected from the group consisting of cephalosporins and aztreonam; and 
 a pH color indicator which changes color when the pH is comprised between 6.4 and 8.4. 
   
     
     
         2 . The kit according to  claim 1 , further comprising a β-lactamase inhibitor. 
     
     
         3 . The kit according to  claim 1 , wherein the reagent kit is comprised in a well or a series of wells of a microtiter plate. 
     
     
         4 . The kit according to  claim 3  wherein the well or series of wells of the microtiter plate further comprise:
 a β-lactamase inhibitor of Ambler class A; 
 a β-lactamase inhibitor of Ambler class B; 
 a β-lactamase inhibitor of Ambler class C; and/or 
 a β-lactamase inhibitor of Ambler class D. 
 
     
     
         5 . The kit according to  claim 1 , wherein the expanded-spectrum β-lactamase substrate is selected from the group consisting of cefotaxime, ceftriaxone, ceftazidime, cefepime, cefpirome and cefotaxime. 
     
     
         6 . The kit according to  claim 1 , wherein the pH color indicator is phenol red.