Pyrosequencing as a Rapid Tool for Identification of GES-Type Extended-Spectrum β-Lactamases

Poirel, Laurent; Naas, Thierry; Nordmann, Patrice

doi:10.1128/jcm.02576-05

Cited by 21 publications

(11 citation statements)

References 23 publications

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“…It is a rapid tool which allows small DNA fragment to be sequenced [114]. It involves a real-time sequencingby-synthesis approach used for SNP detection in genes of CTX-M-type ESBL [115], GES-type ESBL [116], TEM-and SHV-type ESBL [117,118].…”

Section: Pyrosequencingmentioning

confidence: 99%

Detection of expanded-spectrum β-lactamases in Gram-negative bacteria in the 21st century

Al-Bayssari

Dabboussi

Hamzé

et al. 2015

Expert Review of Anti-infective Therapy

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Emerging β-lactamase-producing-bacteria (ESBL, AmpC and carbapenemases) have become a serious problem in our community due to their startling spread worldwide and their ability to cause infections which are difficult to treat. Diagnosis of these β-lactamases is of clinical and epidemiological interest. Over the past 10 years, several methods have been developed aiming to rapidly detect these emerging enzymes, thus preventing their rapid spread. In this review, we describe the range of screening and detection methods (phenotypic, molecular and other) for detecting these β-lactamases but also whole genome sequencing as a tool for detecting the genes encoding these enzymes.

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Section: Pyrosequencingmentioning

confidence: 99%

Detection of expanded-spectrum β-lactamases in Gram-negative bacteria in the 21st century

Al-Bayssari

Dabboussi

Hamzé

et al. 2015

Expert Review of Anti-infective Therapy

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“…Pyrosequencing is suitable for mutation analysis (4,7,8) and has already been used for detecting resistance-conferring genes (11,12,16,21). To our knowledge, this is the first study to describe the use of pyrosequencing as a tool for the rapid detection of aac(6Ј)-Ib-cr and aac(6Ј)-Ib genes.…”

mentioning

confidence: 99%

Rapid Detection of aac(6 ′ )-Ib-cr Quinolone Resistance Gene by Pyrosequencing

et al. 2010

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Pyrosequencing was used to rapidly detect aac(6)-Ib and aac(6)-Ib-cr genes. This plasmid-mediated quinolone resistance determinant is increasing in extended-spectrum beta-lactamase-producing Enterobacteriaceae. This method is faster and more cost-effective than the methods previously described. Sequences obtained with this pyrosequencing method showed 100% concordance with conventional sequencing.

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“…Pyrosequencing has been applied to the detection of mutations in 23S rRNA (6,19) and to the typing of beta-lactamase genes (13,14,16). In the results of our recent study (15), cyclic sequencing of PCRamplified SHV genes did not always reveal an unambiguous sequence.…”

mentioning

confidence: 79%

Typing of SHV Extended-Spectrum β-Lactamases by Pyrosequencing in Klebsiella pneumoniae Strains with Chromosomal SHV β-Lactamase

Haanperä

Forssten

Huovinen³

et al. 2008

Antimicrob Agents Chemother

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In Klebsiella pneumoniae, the cooccurrence of chromosomal and plasmid-mediated beta-lactamases can hinder their accurate molecular detection. We developed a fast and reliable method that allows the typing of isolates carrying more than one SHV gene. The method is based on pyrosequencing the DNA sequence corresponding to amino acid positions 35, 238, and 240.Bacteria producing extended-spectrum beta-lactamases (ESBLs) have become a significant clinical problem, and their detection is problematic (4). Many ESBLs have derived from different beta-lactamases by amino acid substitutions that enable the enzymes to hydrolyze various beta-lactam antibiotics. In SHV, the amino acid substitutions leading to an ESBL phenotype occur at a limited number of positions. Especially, the amino acid substitutions G238S and E240K, according to the numbering of Ambler (2), are important for producing the ESBL phenotype (4, 10).Klebsiella pneumoniae isolates usually carry a chromosomal beta-lactamase, most commonly SHV (3,8), and the plasmid variants have probably derived from the chromosomal SHV genes (5, 9). The chromosomal beta-lactamases may interfere with detection of the ESBL genes usually residing in plasmids; e.g., the PCR-based methods developed for the detection of ESBLs also amplify the chromosomal variants and form a duplex PCR product whose sequence determination by conventional sequencing requires careful analysis (1). The exact SHV type within an isolate harboring more than one SHV gene can be defined by cloning and sequencing.Pyrosequencing is a PCR-based sequencing-by-synthesis method (18) that is ideal for mutation analysis and the detection of heterogeneous sequences (6,7,12). Pyrosequencing has been applied to the detection of mutations in 23S rRNA (6,19) and to the typing of beta-lactamase genes (13,14,16). In the results of our recent study (15), cyclic sequencing of PCRamplified SHV genes did not always reveal an unambiguous sequence. This could be explained by the presence of more than one SHV gene in one strain. The present study was designed to test whether the pyrosequencing technique could be used to resolve these ambiguous SHV sequences. To detect the most-common and important sequences found in SHV ESBLs, the DNA sequences corresponding to the amino acid positions 35, 238, and 240 were included to the pyrosequencing assays (10, 17).We included in this study 40 SHV-positive K. pneumoniae isolates whose SHV gene sequence could not be confirmed by cyclic sequencing due to heterogeneous sequences corresponding to the amino acid positions 35, 238, and/or 240 (15). In addition, 66 K. pneumoniae isolates representing different SHV variants and 10 SHV-positive Escherichia coli isolates were included. The primers used for amplification and pyrosequencing are shown in Table 1. The concentrations of the PCR reagents and temperature-cycling conditions, except for annealing at 54°C and all PCR steps for 30 s, were as described earlier (15).Pyrosequencing was performed by using a PSQ96MA pyrosequencing device and Pyro ...

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Pyrosequencing as a Rapid Tool for Identification of GES-Type Extended-Spectrum β-Lactamases

Cited by 21 publications

References 23 publications

Detection of expanded-spectrum β-lactamases in Gram-negative bacteria in the 21st century

Detection of expanded-spectrum β-lactamases in Gram-negative bacteria in the 21st century

Rapid Detection of aac(6 ′ )-Ib-cr Quinolone Resistance Gene by Pyrosequencing

Typing of SHV Extended-Spectrum β-Lactamases by Pyrosequencing in Klebsiella pneumoniae Strains with Chromosomal SHV β-Lactamase

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