Compositions and methods for rapid identification and phenotypic antimicrobial susceptibility testing of bacteria and fungi

Patent metadata

Field	Value
Publication number	US-11441167-B2
Application number	US-202016950160-A
Country	US
Kind code	B2
Filing date	Nov 17, 2020
Priority date	Nov 20, 2019
Publication date	Sep 13, 2022
Grant date	Sep 13, 2022

Abstract

The present invention relates to compositions and methods for the use of polymerase chain reaction (PCR) as a reporter assay for rapid and simultaneous bacterial identification and phenotype testing for antimicrobial susceptibility (AST). The current invention uses a strategy that has shown the ability for multiplexing and for handling polymicrobial samples for antimicrobial susceptibility testing.

First claim

The invention claimed is:

1. A method to simultaneously identify and determine the antimicrobial susceptibility for one or more antimicrobial(s) of two or more different microorganisms in a polymicrobial biological sample comprising:
a) obtaining the polymicrobial biological sample in which the two or more different microorganisms are believed to be present;
b) optionally culturing the polymicrobial biological sample under conditions that facilitate growth of the two or more different microorganisms and optionally normalizing the cultured polymicrobial biological sample such that the two or more different microorganisms reach a desired concentration;
c) adding the polymicrobial biological sample in two or more reaction wells, whereby the one or more antimicrobial(s) is absent in at least one reaction well, and whereby the one or more antimicrobial(s) is present in one or more reaction wells at one concentration or at more than one varying concentrations, wherein in at least one reaction well, the concentration of the one or more antimicrobial(s) is either the Minimum Inhibitory Concentration (MIC) or the concentration used to classify the two or more different microorganisms as Sensitive, Intermediate or Resistant to the one or more antimicrobial(s);
d) incubating the polymicrobial biological sample in the presence or absence of the one or more antimicrobial(s) for a period of time required to detect inhibition of growth;
e) performing a quantitative 5′ nuclease (TaqMan) real-time PCR assay in each of the two or more reaction wells, whereby, each PCR assay comprises:
(i) an amplifying step with at least a first set of primers and a second set of primers, wherein the first set of primers selectively anneals to a first target gene in a first microorganism and produces a first amplification product if the first microorganism is present in the polymicrobial biological sample, wherein the first microorganism is bacteria in the taxonomic Order Enterobacterales and the first target gene is gyrB, and wherein the second set of primers selectively anneals to a second target gene in a second microorganism and produces a second amplification product if the second microorganism is present in the polymicrobial biological sample, wherein the second microorganism is Acinetobacter baumannii and the second target gene is ompA, and
(ii) a hybridizing step wherein a first TaqMan probe labeled with a first fluorescent dye selectively anneals to the first amplification product and generates a first fluorescent signal and a second TaqMan probe labeled with a second fluorescent dye selectively anneals to the second amplification product and generates a second fluorescent signal;

f) identifying the two or more different microorganisms whereby detection of the first fluorescent signal is indicative of the presence of the first microorganism and detection of the second fluorescent signal is indicative of the presence of the second microorganism; and
g) determining the antimicrobial susceptibility of the two or more different microorganisms by comparing the first and second fluorescent signals detected in the at least one reaction well where the one or more antimicrobial(s) is absent to the first and second fluorescent signals detected in the one or more reaction well(s) where the one or more antimicrobial(s) is present;
wherein steps e), f), and g), are all performed simultaneously in each of the two or more reaction wells.

2. The method of claim 1 wherein the bacteria in the taxonomic Order Enterobacterales is selected from Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella aerogenes, Serratia marcescens , or Proteus mirabilis.

3. The method of claim 2 wherein the bacteria is Klebsiella pneumoniae.

4. The method of claim 1 wherein the first set of primers that selectively anneals to gyrB comprise a forward primer having a nucleotide sequence of SEQ ID NO: 8 and a reverse primer having a nucleotide sequence of SEQ ID NO: 9 and the first TaqMan probe that selectively anneals to the first amplification product of gyrB has a nucleotide sequence of SEQ ID NO: 10.

5. The method of claim 1 wherein the second set of primers that selectively anneals to ompA comprise a forward primer having a nucleotide sequence of SEQ ID NO: 29 and a reverse primer having a nucleotide sequence of SEQ ID NO: 30 and the second TaqMan probe that selectively anneals to the second amplification product of ompA has a nucleotide sequence of SEQ ID NO: 31.

Assignees

• Roche Molecular Systems Inc

Inventors

• Cady Kyle C
• Hanson Brett
• Abdon Paulino
• Chan Ryan
• Lin Patrick
• Mehta Rochak
• Rabang Troy

Classifications

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q2563/107

  fluorescence · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q1/6897

  involving reporter genes operably linked to promoters · CPC title