# US 11,441,167 B2 · Compositions and methods for rapid identification and phenotypic antimicrobial susceptibility testing of bacteria and fungi

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- Number: US 11,441,167 B2
- Document ID: US11441167B2
- Status: Granted
- Kind: B2
- Application number: 16/950,160
- Filed: 2020-11-17
- Published: 2021-05-20
- Granted: 2022-09-13
- Earliest claimed: 2019-11-20
- Assignees: Roche Molecular Systems, Inc.
- Applicants: Roche Molecular Systems, Inc.
- Inventors: Kyle C. Cady; Brett Hanson; Paulino Abdon; Ryan Chan; Patrick Lin; Rochak Mehta; Troy Rabang
- Web page: https://patentgrep.com/patent/US11441167B2

## CPC

- C12Q 1/18: Testing for antimicrobial activity of a material
- C12Q 1/689: for bacteria
- C12Q 1/6895: for plants, fungi or algae
- C12Q 1/6897: involving reporter genes operably linked to promoters
- C12Q 1/6851: Quantitative amplification
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2561/101: Taqman
- C12Q 2561/113: Real time assay
- C12Q 2565/1015: labels being on the same oligonucleotide
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.