Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-28
2001-06-05
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S007200, C435S007400, C435S091100, C435S007320
Reexamination Certificate
active
06242223
ABSTRACT:
BACKGROUND
A disturbing consequence of the use, and over-use, of beta-lactam antibiotics (e.g., penicillins and cephalosporins) has been the development and spread of beta-lactamases. Beta-lactamases are enzymes that open the beta-lactam ring of penicillins, cephalosporins, and related compounds, to inactivate the antibiotic. The production of beta-lactamases is an important mechanism of resistance to beta-lactam antibiotics among Gram-negative bacteria.
Expanded-spectrum cephalosporins have been specifically designed to resist degradation by the older broad-spectrum beta-lactamases such as TEM-1, 2, and SHV-1. Microbial response to the expanded-spectrum cephalosporins has been the production of mutant forms of the older beta-lactamases called extended-spectrum beta-lactamases (ESBLs). Although ESBL-producing Enterobacteriaceae were first reported in Europe in 1983 and 1984, ESBLs have now been found in organisms of diverse genera recovered from patients in all continents except Antarctica. The occurrence of ESBL-producing organisms varies widely with some types more prevalent in Europe (TEM-3), others more prevalent in the United States (TEM-10, TEM-12 and TEM-26), while others appear worldwide (SHV-2 and SHV-5). These enzymes are capable of hydrolyzing the newer cephalosporins and aztreonam. Studies with biochemical and molecular techniques indicate that many ESBLs are derivatives of older TEM-1, TEM-2, or SHV-1 beta-lactamases, some differing from the parent enzyme by one to four amino acid substitutions.
In addition, resistance in
Klebsiella pneumoniae
and
Escherichia coli
to cephamycins and inhibitor compounds such as clavalante have also arisen via acquisition of plasmids containing the chromosomally derived AmpC beta-lactamase, most commonly encoded by
Enterobacter cloacae, Pseudomonas aeruginosa,
and
Citrobacter freundii.
It is of particular concern that genes encoding the beta-lactamases are often located on large plasmids that also contain genes for resistance to other antibiotic classes including aminoglycosides, tetracycline, sulfonamides, trimethoprim, and chloramphenicol. Furthermore there is an increasing tendency for pathogens to produce multiple beta-lactamases. These developments, which occur over a wide range of Gram-negative genera, represent a recent evolutionary development in which common Gram-negative pathogens are availing themselves of increasingly complex repertoires of antibiotic resistance mechanisms. Clinically, this increases the difficulty of identifying effective therapies for infected patients.
Thus, there is a need for techniques that can quickly and accurately identify the types of beta-lactamases that may be present in a clinical isolate or sample, for example. This could have significant implications in the choice of antibiotic necessary to treat a bacterial infection.
SUMMARY OF THE INVENTION
The present invention is directed to the use of oligonucleotide primers specific to nucleic acids characteristic of (typically, genes encoding) certain beta-lactamases. More specifically, the present invention uses primers to identify family specific beta-lactamase nucleic acids (typically, genes) in samples, particularly, in clinical isolates of Gram-negative bacteria. Specific primers of the invention include the primer sequences set forth in SEQ ID NOs: 1-45. As used herein, a nucleic acid characteristic of a beta-lactamase enzyme includes a gene or a portion thereof. A “gene” as used herein, is a segment or fragment of nucleic acid (e.g., a DNA molecule) involved in producing a peptide (e.g., a polypeptide and/or protein). A gene can include regions preceding (upstream) and following (downstream) a coding region (i.e., regulatory elements) as well as intervening sequences (introns, e.g., non-coding regions) between individual coding segments (exons). The term “coding region” is used broadly herein to mean a region capable of being transcribed to form an RNA, the transcribed RNA can be, but need not necessarily be, further processed to yield an mRNA.
Additionally, a method for identifying a beta-lactamase in a clinical sample is provided. Preferably, the clinical sample provided is characterized as a Gram-negative bacteria with resistance to beta-lactam antibiotics. The method includes, providing a pair of oligonucleotide primers, wherein one primer of the pair is complementary to at least a portion of the beta-lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to a different portion of the beta-lactamase nucleic acid in the antisense strand; annealing the primers to the beta-lactamase nucleic acid; simultaneously extending the annealed primers from a 3′ terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein each extension product after separation from the beta-lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair; separating the amplified products; and analyzing the separated amplified products for a region characteristic of the beta-lactamase.
The method, described above, can employ oligonucleotide primers that are specific for nucleic acid of the TEM family of beta-lactamases, the K1 beta-lactamases, the PSE family of beta-lactamases, and the SHV family of beta-lactamases. Additional primers that can be used include those that are specific for nucleic acid of the AmpC beta-lactamases found in
Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Pseudomonas aeruginosa,
and
E. coli.
Still other oligonucleotide primers that are suitable for use in the method of the present invention include primers that are specific for nucleic acid of the plasmid-mediated AmpC beta-lactamases designated as FOX-1, FOX-2, or MOX-1; primers specific for nucleic acid of the OXA-9 beta-lactamase; primers specific for nucleic acid of the OXA-12 beta-lactamase; primers specific for the nucleic acid group of OXA beta-lactamases representing OXA-5, 6, 7, 10, 11, 13, and 14 beta-lactamases; primers specific for the OXA-1 beta-lactamases; and primers specific for nucleic acid of the group of OXA beta-lactamases representing OXA-2, 3, and 15 beta-lactamases.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention is directed to the detection of nucleic acid that is characteristic of (e.g., at least a segment of a gene that codes for) family-specific beta-lactamase nucleic acid in samples (e.g., clinical isolates of Gram-negative bacteria). Specifically, the present invention is directed to the detection of beta-lactamase nucleic acid (preferably, a gene or at least a segment of a gene) using unique primers and the polymerase chain reaction. Using the primers and methods of the present invention, beta-lactamases belonging to Bush groups 1 (AmpC) and 2 (TEM-1, TEM-2, SHV-1, IRTs, K1), for example, can be identified.
The primers and methods of the present invention are useful for a variety of purposes, including, for example, the identification of the primary beta-lactamase(s) responsible for resistance to third generation cephalosporins among Gram-negative bacteria such as
Escherichia coli
and
Klebsiella pneumoniae
(Thomson et al.,
Antimicrob. Agents Chemother
36(9):1877-1882 (1992)). Other sources of beta-lactamases include, for example, a wide range of Enterobacteriaceae, including Enterobacter spp.,
Citrobacter freundii, Morganella morganii,
Providencia spp., and
Serratia marcescens
(Jones,
Diag. Microbiol. Infect. Disease
31(3):461-466 (1998)). Additional beta-lactamase gene sources include
Pseudomonas aeruginosa
(Nordmann et al.,
Antimicrob. Agents Chemother
37(5):962-969 (1993));
Proteus mirabilis
(Bret et al.,
Antimicrob. Agents Chemother
42(5):1110-1114 (1998));
Yersinia enterocolitica
(Barnaud et al.,
FEMS Microbiol. Letters
148(1):15-20 (1997)); and
Klebsiella oxytoca
( Marchese et al.,
Antimicrob. Agents Chemother
42(2):464-467 (1998)).
The methods of the present invention involve the use of the polymerase chain reaction sequ
Ehrhardt Anton F.
Hanson Nancy D.
Sanders Christine C.
Creighton University
Jones W. Gary
Lu Frank
Mueting Raasch & Gebhardt, P.A.
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