# US 5,705,340 A · Methods for screening class B beta-lactamase enzymes from Bacteroides fragilis

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- Number: US 5,705,340 A
- Document ID: US5705340A
- Status: Granted
- Kind: A
- Application number: 08/459,264
- Filed: 1995-06-02
- Granted: 1998-01-06
- Earliest claimed: 1990-07-16
- Assignees: American Cyanamid Company
- Inventors: Beth A. Rasmussen; Francis P. Tally; Yakov Gluzman
- Web page: https://patentgrep.com/patent/US5705340A

## CPC

- C12N 9/86: acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)}
- C12Q 1/18: Testing for antimicrobial activity of a material
- G01N 2333/986: acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

This invention is directed to the elucidation of DNA sequences encoding Class B beta-lactamase enzymes from Bacteroides fragilis and the amino acid sequences of those enzymes. This invention is also directed to screening methods for identifying antibiotics which are resistant to such beta-lactamase enzymes or for identifying compounds which inactivate such beta-lactamase enzymes.

## Claims

### Claim 1

A method of screening for beta-lactam antibiotics which are resistant to a Class B beta-lactamase enzyme of Bacteroides fragilis, which comprises:
(a) inserting a plasmid containing a DNA sequence encoding a beta-lactamase enzyme of Bacteroides fragilis into a suitable bacterial host organism which expresses the Class B enzyme;
(b) adding a beta-lactam antibiotic to the host organism; and
(c) observing the inhibition of growth of the organism by the antibiotic, such that if growth is inhibited at low concentrations of the antibiotic, then the antibiotic is resistant to a Class B beta-lactamase enzyme of Bacteroides fragilis.

### Claim 2

A method of screening for beta-lactam antibiotics which are resistant to a Class B beta-lactamase enzyme of Bacteroides fragilis, which comprises:
(a) inserting a plasmid containing a DNA sequence encoding a beta-lactamase enzyme of Bacteroides fragilis into a Bacteroides organism;
(b) adding a beta-lactam antibiotic to the Bacteroides organism; and
(c) observing the inhibition of growth of the organism by the antibiotic, such that if growth is inhibited at low concentrations of the antibiotic, then the antibiotic is resistant to a Class B beta-lactamase enzyme of Bacteroides fragilis.

### Claim 3

A method of screening for compounds which inactivate a Class B beta-lactamase enzyme of Bacteroides fragilis, which comprises:
(a) inserting a plasmid containing a DNA sequence encoding a beta-lactamase enzyme of Bacteroides fragilis into a suitable bacterial host organism which expresses the Class B enzyme;
(b) adding to the host organism a compound together with a beta-lactam antibiotic known to be inactivated by the enzyme; and
(c) observing the inhibition of growth of the organism by the antibiotic, such that if growth is inhibited at low concentrations of the antibiotic, the compound inactivates a Class B beta-lactamase enzyme of Bacteroides fragilis.

### Claim 4

A method of screening for compounds which inactivate a Class B beta-lactamase enzyme of Bacteroides fragilis, which comprises:
(a) inserting a plasmid containing a DNA sequence encoding a beta-lactamase enzyme of Bacteroides fragilis into a Bacteroides organism;
(b) adding to the Bacteroides organism a compound together with a beta-lactam antibiotic known to be inactivated by the enzyme; and
(c) observing the inhibition of growth of the organism by the antibiotic, such that if growth is inhibited at low concentrations of the antibiotic, the compound inactivates a Class B beta-lactamase enzyme of Bacteroides fragilis.