Automated method for analyzing and interpreting an antimicrobial susceptibility

Patent metadata

Field	Value
Publication number	US-10837044-B2
Application number	US-201515516094-A
Country	US
Kind code	B2
Filing date	Oct 5, 2015
Priority date	Oct 6, 2014
Publication date	Nov 17, 2020
Grant date	Nov 17, 2020

Abstract

The present invention relates to a method of interpreting different antibiogram images in which it is possible to recognize a phenotype of bacterial resistance relative to antibiotics by comparing photographs using a photographic image bank of the reference antibiogram without any need to interpret them using the EUCAST or CA-SFM interpretation data; providing that for a given phenotype, there is available a collection of photographs of a plurality of bacteria of the same species and of the same phenotype.

First claim

The invention claimed is:

1. A method of automatically analyzing and interpreting an antibiogram of a sample of a microorganism for analysis, the method serving to determine a phenotype of resistance or sensitivity to at least one antimicrobial compound, said phenotype being selected from a plurality of different reference phenotypes for each of the reference species of microorganism in a plurality of different species of reference microorganisms, wherein:
a) the antibiogram of the sample is prepared using a methodology on a solid culture medium, which methodology comprises:
depositing a concentration of microorganism of the sample of microorganism, on said solid culture medium;
depositing a number n of a plurality of substrates on said solid culture medium, the substrates containing a concentration of antimicrobial compound(s) and suitable for diffusing one or more different antimicrobial compounds for each of the n substrates;
incubating the sample of microorganism deposited on said solid culture medium under conditions and for a duration;
b) acquiring a photographic image of the antibiogram of the sample obtained in step a); and
c) determining said phenotype of the sample of microorganism, by using software to compare the photographic image obtained in step b) with reference images of reference antibiograms in a database,
wherein said database comprises a plurality of at least p different reference images of antibiograms of p different strains of the same microorganism for each reference phenotype of each reference species of microorganism.

2. The method according to claim 1 , further comprising preparing all of the reference antibiograms in step c) using the same said methodology as the antibiogram of the sample, and taking said reference images and the image for analysis with the same photographic image capture apparatus and under the same conditions comprising a same distance, a same brightness, a same background, and a same resolution.

3. The method according to claim 1 , wherein a plurality, n, of said substrates is used, where n lies in the range 5 to 20, and said database comprises a plurality of at least p different reference images of antibiograms of p different strains of the same microorganism, where p is not less than 5.

4. The method according to claim 3 , wherein n lies in the range 6 to 16, and wherein p is not less than 10.

5. The method according to claim 1 , wherein, in step c), said software is used to compare the shape of the outline of the rings corresponding to inhibition zones around the substrates.

6. The method according to claim 5 , wherein said substrates are in the form of wafers, and said software recognizes at least one of antibiotic code letters and dosage numerals specified on the wafers.

7. The method according to claim 1 , wherein said microorganism is a bacterium, wherein a minimum acceptable similarity percentage between an image and a reference image is determined, referred to here as a similarity threshold, and wherein the bacterium of the sample is determined as presenting said reference phenotype of said reference bacterium if the recognition software evaluates a similarity percentage between the image of the antibiogram of the sample and a reference image corresponding to said reference phenotype of said reference species of bacterium that is not less than a minimum acceptable threshold similarity percentage.

8. The method according to claim 7 , wherein the minimum acceptable threshold similarity percentage is at least 70%.

9. The method according to claim 1 , wherein said microorganism is a bacterium, wherein a minimum threshold similarity percentage between said different reference images of different antibiograms of p different strains for each reference phenotype of each reference species of bacterium is determined, referred to here as the reference similarity threshold, wherein said reference similarity threshold percentage corresponding to the lowest similarity percentage between the two most different reference images for each reference phenotype of each reference species of bacteria is determined, and wherein the bacterium of the sample is determined as presenting said reference phenotype of said reference bacterium if the recognition software evaluates a similarity percentage that is not less than said reference similarity threshold between the image of the antibiogram of the sample and a reference image corresponding to said reference phenotype of said reference species of bacterium.

10. The method according to claim 1 , wherein said reference phenotype is a resistance or sensitivity classification for a given antibiotic or group of antibiotics.

11. The method according to claim 10 , wherein said reference phenotype includes a synergy phenotype between two antibiotics corresponding to two antibiotics for which the reference microorganism is resistant when they are taken separately and for which the reference microorganism is sensitive when they are taken in association.

12. The method according to claim 1 , wherein the reference phenotype is a resistance or sensitivity classification for a plurality of antibiotics and/or for a group of antibiotics.

13. The method according to claim 1 , wherein said reference species of microorganism is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Klebsiella oxytoca, Enterobacter cloacea, Enterobacter aerogenes, Entercoccus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus , and Staphylococcus epidermidis.

14. The method according to claim 1 , wherein the antibiogram of the sample is made using wafers comprising the following antibiotics and with the following marking corresponding to said antibiotics: AMX for amoxicillin; AMC for amoxicillin-clavulanic acid; GEN for gentamicin; SXT for trimethoprim-sulfamethoxazole (cotrimoxazole); TIC for ticarcillin; TCC for ticarcillin-clavulanic acid; TZP for piperacillin-tazobactam; CAZ for ceftazidime; IMP for imipenem; COL or CL for colistin; FOX for cefoxitine; VAN or VA for vancomycin; TEC for teicoplanin; CLI or DA for clindamycin; CRO for ceftriaxone; FEP for cefepime; L or LIN for lincomycin; FUR or NT for nitrofurantoin; CIP for ciprofloxacin; OFX for ofloxacin; CTX for cefotaxime; AK or AN for amikacin; TOB or TM for tobramycin; FOX for cefoxitine; ATM for aztreonam; ERT for ertapenem; FF or FOS for fosfomycin; DOI for doxycycline; K for kanamycin; L or LIN for lincomycin; LNZ for linezolid; NA for nalidixic acid; NOR for norfloxacin; MET for metronidazole; MEM for meropenem; PT for pristinamycin; P for penicillin G; RA for rifampicin; TGC for tigecycline; TEL for telithromycin; E for erythromycin; and OX or OXA for oxacillin.

15. The method according to claim 14 , wherein the antibiogram under analysis and/or said database comprises the reference phenotypes for the reference species of bacteria corresponding to the following inhibitors/resistances (I/R) or sensitivities (S) relative to concentrations of six antibiotic compounds selected from the following groups a1) to a6), arranged in a circle in the following order for each group:
a1) AMX, AMC, TCC, CRO, FEP, and IMP,
a2) AMX, AMC, TZP, CRO, FEP, and IMP,
a3) AMC, TZP, CRO, FEP, IMP, and COL,
a4) AMX, GEN, LIN, FUR, VAN, and TEC,
a5) TIC, TCC, TZP, FEP, CAZ, and IMP, and
a6) FOX, CLI, SXT, GEN, VAN, and TEC.

16. The method according to claim 14 , wherein the antibiogram under analysis and/or said database comprises the following reference phenotypes for the following reference species of bacteria corresponding to the following inhibitors/resistances

Assignees

• Fond Mediterranee Infection
• Univ Aix Marseille
• Assist Publique—Hopitaux De Marseille
• Centre Nat Rech Scient
• Inst Nat Sante Rech Med

Inventors

• Rolain Jean-Marc
• Raoult Didier
• Le Page Stéphanie
• Buffet Sylvain

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

• C12Q1/18

  Testing for antimicrobial activity of a material · CPC title

• G06T2207/30024

  Cell structures in vitro; Tissue sections in vitro · CPC title

• C12Q1/20 Primary

  using multifield media · CPC title

• G06T7/0014

  using an image reference approach · CPC title