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Antimicrobial Agents and Chemotherapy, March 2009, p. 977-986, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01155-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Pyrosequencing Using the Single-Nucleotide Polymorphism Protocol for Rapid Determination of TEM- and SHV-Type Extended-Spectrum β-Lactamases in Clinical Isolates and Identification of the Novel β-Lactamase Genes blaSHV-48, blaSHV-105, and blaTEM-155{triangledown}

C. Hal Jones,*{dagger} Alexey Ruzin,{dagger} Margareta Tuckman, Melissa A. Visalli, Peter J. Petersen, and Patricia A. Bradford

Infectious Diseases Discovery Research, Wyeth Research, Pearl River, New York

Received 28 August 2008/ Returned for modification 19 October 2008/ Accepted 8 December 2008

TEM- and SHV-type extended-spectrum β-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the blaTEM and blaSHV genes. Three novel β-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing blaSHV-105 were >128, 128, and >128 µg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing blaTEM-155 were >128, 64, and > 128 µg/ml, respectively. Pyrosequence analysis determined the true identity of the β-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.

* Corresponding author. Mailing address: Wyeth Research, 401 N. Middletown Rd., Bldg. 200, Rm. 3219, Pearl River, NY 10965. Phone: (845) 602-4612. Fax: (845) 602-5671. E-mail: jonesh3{at}wyeth.com

{triangledown} Published ahead of print on 15 December 2008.

{dagger} The first two authors contributed equally to the work described here.

Antimicrobial Agents and Chemotherapy, March 2009, p. 977-986, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01155-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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