Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-18
2004-04-13
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S440000
Reexamination Certificate
active
06720142
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of predicting the evolutionary potential of a mutant resistance gene, the resulting mutant resistant genes and their expression products, as well as a method of screening a candidate drug for activity against a pathogen including a mutant resistance gene and a method of assessing the potential longevity of a candidate drug.
BACKGROUND OF THE INVENTION
Antibiotics have proven to be one of medicine's most effective tools in combating disease, but their utility is constantly being challenged by the emergence of antibiotic-resistant target organisms and their future effectiveness is now in doubt. Because of their efficiency, specificity, and general absence of toxicity, &bgr;-lactam antibiotics account for about 50% of global antibiotic consumption (Livermore, “Are all beta-lactams Created Equal?”
Scand. J. Infecl. Dis. SuDDI.
101: 33-43 (1996); Matagne et al., “Catalytic Properties of Class A beta-lactamases: Efficiency and Diversity,”
Biochem. J.
330:581-598 (1998)). Since the clinical introduction of benzylpenicillin about 50 years ago the efficiency of &bgr;-lactams has been continuously challenged by the emergence of resistant pathogens. As a result new molecules have been progressively introduced with modifications that are increasingly different from the original penicillin (Matagne et al., “Catalytic Properties of Class A beta-lactamases: Efficiency and Diversity,”
Biochem. J.
330:581-598 (1998)). Genes for resistance to &bgr;-lactams are typically plasmid-borne and encode enzymes, called &bgr;-lactamases, that degrade and inactivate &bgr;-lactam antibiotics. Among plasmid-borne resistance genes the TEM-1 &bgr;-lactamase is the most prevalent, accounting for 75% of the &bgr;-lactamase in Gram negative organisms worldwide (Amyes, “Genes and Spectrum: The Theoretical Limits,”
Clin. Infect. Dis.
27 Suppl 1:S21-28 (1998)). The success of TEM-1 &bgr;-lactamase and its relatives SHV-1, TEM-2 and OXA-1 is partly the result of its location on plasmids with insertion elements that permitted very rapid dissemination of the &bgr;-lactamase genes, and partly the result of continued evolution of the enzyme itself in response to the introduction of new drugs, particularly the cephalosporins and extended-spectrum &bgr;-lactams known as third generation cephalosporins.
Another group of &bgr;-lactamases, the Class C &bgr;-lactamases, are generally quite active toward cephalosporins, including the third generation derivatives, but have not been taken as a serious threat until recently, because the genes for Class C &bgr;-lactamases are typically located on chromosomes rather than on plasmids. The chromosomal ampC genes are found in a variety of Gram negative bacteria, including both the Enterobacteriaceae and Pseudomonas species. The ampC genes are typically expressed at a low level, as in
E. coli,
or are inducible by penicillins and early generation cephalosporins but not inducible by third and fourth generation cephalosporins. Over the last decade, however, it has been found that Gram negative pathogens that are hyper-producers of ampC &bgr;-lactamases are resistant to all but a few of the most recently introduced &bgr;-lactam antibiotics (Livermore, “Are all beta-lactams Created Equal?”
Scand. J. Infect. Dis. Suppl.
101: 33-43 (1996)). By now 25-50% of Enterobacter isolates from ICU patients in many major Western and Far Eastern hospitals are AmpC hyper-producers and are resistant to all penicillins and cephalosporins except imipenem, meropenem, and temicillin (Livermore, “Are all beta-lactams Created Equal?”
Scand. J. Infect. Dis. Suppl.
101: 33-43 (1996)). Even more worrying are several recent reports of derepressed ampC genes located on plasmids found in pathogenic Gram negative bacteria (Bauernfeind et al., “Characterization of the Plasmidic beta-lactamase CMY-2, which is Responsible for Cepharnycin Resistance,”
Antimicrob. Agents Chemother.
40:221-224 (1996); Horii et al., “Characterization of a Plasmid-borne and Constitutively Expressed blaMOX-1 Gene Encoding AmpC-type beta-lactamase,”
Gene
139:93-98 (1994); Jacoby et al., “More Extended-spectrum beta-lactamases,”
Antimicrob. Agents Chemother.
35:1697-1704 (1991); Papanicolaou et al., “Novel Plasmid-mediated beta-lactamase (MIR-1) Conferring Resistance to Oxyimino- and alpha-methoxy beta-lactams in Clinical Isolates of
Klebsiella pneumoniae,” Antimicrob. Agents Chemother.
34:2200-2209 (1990)). Although Aymes (“Genes and Spectrum: The Theoretical Limits,”
Clin. Infect. Dis.
27 Suppl 1:S21-28 (1998)) suggests that AmpC &bgr;-lactamases at present do not seem efficient enough to cause widespread clinical problems, others (Lindberg et al., “Contribution of Chromosomal beta-lactamases to beta-lactam Resistance in Enterobacteria,”
Rev. Infect. Dis.
8 Suppl 3:S292-304 (1986); Morosini et al., “An Extended-spectrum AmpC-type beta-lactamase Obtained by in vitro Antibiotic Selection,”
FEMS Microbiol. Lett.
165:85-90 (1998); Pitout et al., “Antimicrobial Resistance with Focus on beta-lactam Resistance in Gram-negative Bacilli,”
Am. J. Med.
103:51-59 (1997)) are quite concerned that the AmpC &bgr;-lactamases constitute a pool from which clinically significant resistant strains may well emerge. That concern is exacerbated by the finding that in a clinical isolate of
Enterobacter cloacae
the AmpC &bgr;-lactamase has extended its substrate range to include the oxyimino &bgr;-lactams as the result of a tandem duplication of three amino acids (Nukaga et al., “Molecular Evolution of a Class C beta-lactamase Extending its Substrate Specificity,”
J. Biol. Chem.
270:5729-5735 (1995)). More recently, Morosini and his colleagues (Morosini et al., “An Extended-spectrum AmpC-type beta-lactamase Obtained by in vitro Antibiotic Selection,”
FEMS Microbiol. Lett.
165:85-90 (1998)) applied direct selection to isolate a mutant of an
Enterobacter cloacae
AmpC &bgr;-lactamase in which the activity toward a fourth generation cephalosporin had increased 267 fold as the result of a single amino acid replacement. Together, the fact that mutations to hyper-expression of AmpC occur readily and the facts that hyper-expressed ampC &bgr;-lactamases already confer resistance to penicillins and third generation cephalosporins, are moving onto plasmids, and can easily mutate to significant activity against fourth generation cephalosporins suggest that AmpC &bgr;-lactamases may very well constitute a potentially serious clinical threat.
Similar development of resistance can be seen in other antibiotic resistance genes such as the katG, rpoB, and rpsL genes of
Mycobacterium tuberculosis
which have resulted in multi drug resistance to isoniazid, rifampicin and streptomycin (C.D.C., “Outbreak of Multidrug-resistant Tuberculosis—Texas, California, and Pennsylvania,”
MMWR
39:369-372 (1990); C.D.C., “Nosocomial Transmission of Multidrug-resistant Tuberculosis Among HIV-infected Persons—Florida and New York 1988-1991,”
MMWR
40:585-591 (1991); C.D.C., “Transmission of Multidrug-resistant Tuberculosis from an HEV-positive Client in a Residential Substance Abuse Treatment Facility—Michigan,”
MMWR
40:129-131 (1991)); adaptive virus resistance to antiviral agents as seen, for example, with HIV-encoded protease resistance to the protease inhibitors saquinavir, ritonavir, and indinavir (Condra et al., “In vivo Emergence of HIV-1 Variants Resistant to Multiple Protease Inhibitors,”
Nature
374:569-571 (1995)); antifungal resistance genes such as the atrc multiple drug resistance gene of
Aspergillus nidulans
(U.S. Pat. No. 6,060,264 to Skatrud et al.) and pdr1 multidrug resistance gene of
Saccharomyces cerevisiae
(Balzi et al., “Me Multidrug Resistance Gene pdr1 from
Saccharomyces cerevisiae,” J. Biol. Chem.
262(35):16871-16879 (1987)); and the antimalarial resistance genes such as the pfmdrl gene of
Plasmodium falciparum
(Ruetz et al., “The pfmdrl Gene of
Plasmodium falciparum
Confers Cellular Resistance to Antimalarial Drugs in Yeast Cells,”
Proc
Horlick Kenneth R.
Nixon & Peabody LLP
University of Rochester
LandOfFree
Method of determining evolutionary potential of mutant... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of determining evolutionary potential of mutant..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of determining evolutionary potential of mutant... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3220267