Methods for detecting beta-lactamase-producing bacteria

Patent metadata

Field	Value
Publication number	US-10718006-B2
Application number	US-201615562845-A
Country	US
Kind code	B2
Filing date	Apr 1, 2016
Priority date	Apr 3, 2015
Publication date	Jul 21, 2020
Grant date	Jul 21, 2020

Abstract

An electrochemical method is provided for in-vitro determination of the presence of bacteria producing lactamases, in a sample that may contain such bacteria.

First claim

The invention claimed is:

1. A method for in-vitro determination of the presence of bacteria producing beta-lactamases, in a sample, said method comprising the following steps:
(a) incubating said sample in a medium containing a substrate of the beta-lactamases, said substrate having electrochemical properties, said substrate being nitrocefin or the compound HMRZ-86 (E isomer of (6R,7R)-trifluoroacetate 7-[[2-(2-amino-4-thiazolyl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-1-aza-3-[2-(2,4-dinitrophenyl)ethenyl]-8-oxo-5-thiabicyclo[4.2.0]oct-2-ene-2-carboxylic acid),
(b) applying an amperometric measurement, comprising a cyclic voltammetric method, by using a working electrode based on carbon or based on a noble metal or based on metal oxide, to the aforesaid medium obtained at the end of step (a) and to a negative control, respectively,
(c) measuring the difference of value of the intensity of the anodic current corresponding to oxidation of the hydrolyzed substrate, and that of the negative control.

2. The method according to claim 1 , characterized in that said method comprises the following steps:
(a) incubating the sample in a medium containing a substrate of the beta-lactamases, the substrate having amperometric properties;
(b) applying an amperometric measurement to the aforesaid medium obtained at the end of step (a) and to a negative control, respectively; and
(c) determining the presence of the beta-lactamase-producing bacteria by measuring the difference between the value of the intensity of the anodic current corresponding to oxidation of the hydrolyzed substrate, measured for the aforesaid medium obtained at the end of step (a), and the value of the intensity of the anodic current measured for the negative control.

3. The method according to claim 2 , said method additionally comprising, after step (c), step (d) consisting of measuring the difference between the value of the intensity of the anodic current measured for said medium obtained at the end of step (a) and a calibration curve established under the same conditions.

4. The method according to claim 1 making it possible in addition to distinguish the type of beta-lactamases selected from penicillinases, extended-spectrum beta-lactamases, inducible cephalosporinases, hyperproduced cephalosporinases and carbapenemases, produced by the aforesaid bacteria in a sample that may contain them, said method comprising the following steps:
(a) incubating, in parallel, a fraction of the aforesaid sample in:
a culture medium A, a culture medium B and a culture medium C:
culture medium A being a basic culture medium,
culture medium B being a basic culture medium supplemented with a third-generation cephalosporin, and
culture medium C being a basic culture medium supplemented with a cephalosporin and a penicillinase inhibitor, and

optionally in a culture medium B′, a culture medium C′, a culture medium D, a culture medium E:
culture medium B′ being a basic culture medium supplemented with a third-generation cephalosporin different from that present in medium B;
culture medium C′ being culture medium B′ supplemented with a penicillinase inhibitor;
culture medium D being a basic culture medium supplemented with a cephalosporin and a cephalosporinase inhibitor, and
culture medium E being a basic culture medium supplemented with a carbapenem,

(b) incubating, in parallel, the aforesaid culture media obtained at the end of the incubation in step (a) in a medium containing nitrocefin; and
(c) applying an amperometric means, to the aforesaid media obtained at the end of step (b) and thereby determining the presence of beta-lactamase-producing bacteria and distinguishing the type of beta-lactamases.

5. The method according to claim 4 in order to determine the presence of the bacteria producing an extended-spectrum beta-lactamase, said method comprising the following steps:
(a) incubating, in parallel, a fraction of the aforesaid sample in a culture medium A, a culture medium B and a culture medium C, and optionally in a culture medium B′ and a culture medium C′, as defined according to claim 4 ;
(b) incubating, in parallel, the aforesaid culture media obtained at the end of the incubation in step (a) in a medium containing nitrocefin; and
(c) applying an amperometric means, to the aforesaid media obtained at the end of step (b) in order to determine the presence of the bacteria producing an extended-spectrum beta-lactamase.

6. The method according to claim 4 , in order to determine and distinguish the beta-lactamase-producing bacteria in a sample that may contain them, comprising the following steps:
(a) incubating, in parallel, a fraction of the aforesaid sample in a culture medium A, a culture medium B, a culture medium C, a culture medium D, a culture medium E, optionally a medium B′ and a medium C′, as defined according to claim 4 ;
(b) incubating, in parallel, the aforesaid culture media obtained at the end of the incubation in step (a) in a medium containing nitrocefin;
(c) applying an amperometric measurement to the aforesaid media obtained at the end of step (b) and to a negative control, respectively;
(d) determining the presence of beta-lactamase-producing bacteria in said sample by comparing the value of the intensity of the anodic current corresponding to the oxidation of hydrolyzed nitrocefin obtained for the fraction cultured in culture medium A with the value of the intensity of the current obtained for the negative control; and
(e) distinguishing the type of beta-lactamases produced by the aforesaid bacteria in said sample, by measuring the respective difference between the values of the intensity of the aforesaid anodic current obtained for the fractions cultured in parallel in culture media A, B, C, D and E, optionally B′ and C′ and the respective values obtained for a reference bacterial strain.

7. The method according to claim 6 , further comprising a step (f) after step (e) making it possible to quantify beta-lactamase-producing bacteria distinguished in step (e) by measuring the difference between the value of the intensity of the anodic current corresponding to nitrocefin hydrolyzed by said beta-lactamase and a calibration curve established under the same conditions.

8. The method according to claim 4 , for distinguishing the type of beta-lactamases and optionally defining the subfamily of carbapenemases produced by the aforesaid bacteria in a sample that may contain them, said method comprising the following steps:
(a) incubating, in parallel, a fraction of the aforesaid sample in a culture medium A, a culture medium B, a culture medium C, a culture medium D, a culture medium E, and a culture medium F, and optionally a culture medium B′ and a culture medium C′;
the media A, B, B′, C, C′, D, E being as defined according to claim 4 ;
culture medium F being a basic culture medium supplemented with a carbapenem and an inhibitor specific to a subfamily of carbapenemases;
(b) incubating, in parallel, the aforesaid culture media obtained at the end of the incubation in step (a) in a medium containing nitrocefin;
(c) applying an amperometric measurement to the aforesaid media obtained at the end of step (b) and to a negative control, respectively;
(d) determining the presence of beta-lactamase-producing bacteria in said sample by comparing the value of the intensity of the anodic current corresponding to the oxidation of hydrolyzed nitrocefin obtained for the fraction cultured in culture medium A with the value of the intensity of the current obtained for the negative control; and
(e) distinguishing the type of beta-lactamases produced by the aforesaid bacteria in said sample, by measuring the respective differences between values of the intensity

Assignees

• Univ Bourgogne
• Centre Hospitalier Regional Univ De Dijon
• Institut National De La Rech Agronomique—Inra
• Institut National De La Rech Agronmique

Inventors

• Dequaire-Rochelet Murielle
• Hartmann Alain
• Neuwirth Catherine
• Chantemesse Benoit

Classifications

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• G01N27/48

  using polarography, i.e. measuring changes in current under a slowly-varying voltage · CPC title

• C12Y305/02006

  Beta-lactamase (3.5.2.6) · CPC title

• G01N27/3277

  being a redox reaction, e.g. detection by cyclic voltammetry (voltammetry per se G01N27/42, G01N27/48) · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title