title: Pyrosequencing using the single-nucleotide polymorphism protocol for rapid determination of TEM- and SHV-type extended-spectrum beta-lactamases in clinical isolates and identification of the novel beta-lactamase genes blaSHV-48, blaSHV-105, and blaTEM-155.
abstract: TEM- and SHV-type extended-spectrum beta-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 bla(TEM) and bla(SHV) genes. Three novel beta-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 bla(SHV-105) were >128, 128, and >128 microg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing bla(TEM-155) were >128, 64, and > 128 microg/ml, respectively. Pyrosequence analysis determined the true identity of the beta-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.
authors.affiliation: Infectious Diseases Discovery Research, Wyeth Research, Pearl River, New York 10965, USA. jonesh3@wyeth.com
authors.forename: C Hal
authors.initials: CH
authors.lastname: Jones
authors.forename: Alexey
authors.initials: A
authors.lastname: Ruzin
authors.forename: Margareta
authors.initials: M
authors.lastname: Tuckman
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Visalli
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Petersen
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Bradford
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D013997:Time Factors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1