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Patent Issued for Compositions and Methods for the Identification of a Carbapenemase Gene

The patent's inventors are Whiteford, Craig C. (York, PA); Yu, Charles (Lutherville, MD).

This patent was filed on May 16, 2011 and was cleared and issued on December 4, 2012.

From the background information supplied by the inventors, news correspondents obtained the following quote: "The Enterobacteriaceae are a large family of bacteria, including many of the more familiar pathogens, such as Salmonella and Escherichia coli. Members of genera belonging to the Enterobacteriaceae family have earned a reputation placing them among the most pathogenic and most often encountered organisms in clinical microbiology. These large gram-negative rods are usually associated with intestinal infections but can be found in almost all natural habitats. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. Escherichia coli, better known as E. coli, is one of the most important model organisms, and its genetics and biochemistry have been closely studied.

"Klebsiella pneumoniae is a gram-negative, nonmotile, encapsulated, lactose-fermenting, facultatively anaerobic bacterium found in the normal flora of the mouth, skin, and intestines. It is clinically the most important member of the Klebsiella genus of Enterobacteriaceae. K. pneumoniae can cause bacterial pneumonia, though it is more commonly implicated in hospital-acquired urinary tract and wound infections, particularly in immunocompromised individuals. Klebsiella ranks second to E. coli for urinary tract infections in older persons. It is also an opportunistic pathogen for patients with chronic pulmonary disease, enteric pathogenicity, nasal mucosa atrophy, and rhinoscleroma. Feces are the most significant source of patient infection, followed by contact with contaminated instruments. K. pneumoniae is an increasingly nosocomial infection as antibiotic resistant strains continue to appear.

"Klebsiella possesses a chromosomal class A beta-lactamase giving it inherent resistance to ampicillin. Many strains have acquired an extended-spectrum beta-lactamase (ESBL) with additional resistance to carbenicillin, ampicillin, quinolones, and increasingly to ceftazidime. Carbapenem antibiotics have been important agents for the management of gram-negative infections, particularly when caused by difficult nosocomial pathogens.

"Carbapenems have the broadest activity spectra of any beta-lactam antibiotic and are often the most appropriate agents for use in the treatment of infections caused by multiresistant gram-negative bacteria. Carbapenems are considered to be the agents of choice for the treatment of infections due to Enterobacteriaceae possessing extended-spectrum beta-lactamases (ESBLs). The prevalence of ESBL-producing Klebsiella pneumoniae has been rising in the United States, and is approaching 50% of isolates in some regions. When such high rates of ESBL-producing organisms are encountered, carbapenems become an increasingly important therapeutic option. Over the past few years, a progressive increase in carbapenem-resistant gram-negative bacteria has been observed in some areas. In the United States, carbapenem resistance has been largely attributed to expression of a class C cephalosporinase and loss of outer membrane porins in isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and rarely, K. pneumoniae. Carbapenem-hydrolysing beta-lactamases (carbapenemases) have been rarely recovered in K. pneumoniae. However, isolates possessing carbapenemases KPC-1, KPC-2, and KPC-3 have been recently identified in the northeastern United States. These isolates are often resistant to multiple antibiotic classes, presenting clinicians with very limited therapeutic options.

"The emergence of highly resistant organisms causing outbreaks of infections is a significant problem that the microbiology and infectious disease community have been dealing with for several years. Now, the emergence of carbapenem-resistant Klebsiella pneumoniae can be added to the growing list of highly resistant organisms. An outbreak of carbapenem-resistant K. pneumoniae infections that occurred in multiple hospitals in New York City in 2005 brought widespread attention to these organisms.

"KPC enzymes are beta-lactamases that mediate resistance to extended-spectrum cephalosporins as well as resistance to the carbapenems. These carbapenemases were first reported in 2001 in North Carolina but have now been isolated in various parts of the United States, most frequently on the East coast. Detection of isolates that produce a carbapenemase is important for better management of therapy and for infection control."

Supplementing the background information on this patent, NewsRx reporters also obtained the inventors' summary information for this patent: "Compositions and methods for the rapid and sensitive detection of a carbapenemase gene that confers antibiotic resistance are provided. The compositions comprise oligonucleotide novel primer and probe sets for use in detecting the presence of this gene in a sample. These primers and probe sets can be used in amplification methods (such as PCR, particularly quantitative PCR) and packaged into kits for use in amplification methods for the purpose of detecting the presence of a carbapenemase gene in a test sample, particularly a patient sample, whereby detection of the gene is indicative that the sample comprises a bacterium that is resistant to carbapenems.

"Thus, in one embodiment, the present invention provides for novel oligonucleotide primers set forth in SEQ ID NOs:1, 2, 4, 5, 7, 8, 14, 15, 17, 18, and 20 and the novel oligonucleotide probe sequences set forth in SEQ ID NOs:3, 6, 9, 16, and 19. These sequences can be used in a method of detecting a carbapenemase gene in a sample, the presence of which is indicative that the sample comprises a bacterium having carbapenem resistance.

"Further provided are kits useful for the detection of a carbapenemase gene in a sample, where the kits comprise a composition according to the present invention. The kits may further comprise instructions for using the provided composition in a polymerase-based amplification reaction, for example, PCR or QPCR.

"In another embodiment, the present invention relates to a method of detecting a carbapenemase in a sample using polymerase-based amplification of a target nucleic acid region present in the bacteria, the method comprising: (a) providing a test sample suspected of containing an enterobacterium having carbapenem resistance, (b) contacting the sample with a composition of the invention under conditions sufficient to provide polymerase-based nucleic acid amplification products comprising a target nucleic acid region of a nucleotide sequence encoding a carbapenemase; and © detecting the presence of the nucleic acid amplification products as an indication of the presence of a carbapenemase in the test sample. In various embodiments, the test sample is a direct sample, and the methods and compositions of the invention are capable of detecting the presence of carbapenemase in the direct sample at a concentration of bacteria that is within the range of bacterial load typically found in a sample collected from a subject infected with that bacterium.

"The present invention also relates to use of the primers according to the present invention, wherein the primers or probes have the sequences according to any of the sequences as defined in SEQ ID NOS:1-9 and 14-20 and 14-20."

For the URL and additional information on this patent, see: Whiteford, Craig C.; Yu, Charles. Compositions and Methods for the Identification of a Carbapenemase Gene. U.S. Patent Number 8324364, filed May 16, 2011, and issued December 4, 2012. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser'Sect1=PTO2&Sect2=HITOFF&p=71&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=3509&f=G&l=50&co1=AND&d=PTXT&s1=20121204.PD.&OS=ISD/20121204&RS=ISD/20121204

Keywords for this news article include: Polymerase, Carbapenems, Amidohydrolases, beta-Lactamases, Sulfur Compounds, Enzymes and Coenzymes, Becton Dickinson and Company.

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