Dataset Information

Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum ?-lactamases (ESBL).

ABSTRACT: The current global spread of multi-resistant Gram-negatives, particularly extended spectrum ?-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-?-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-?-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other ?-lactamase families.

SUBMITTER: Stein C

PROVIDER: S-EPMC3818264 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).

Stein Claudia C Makarewicz Oliwia O Pfeifer Yvonne Y Brandt Christian C Ramos João Costa JC Klinger Mareike M Pletz Mathias W MW

PloS one 20131105 11

The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast mo ...[more]

PMID: 24224038

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Project description:CTX-M-type extended-spectrum beta-lactamases (ESBLs) have become increasingly common worldwide, with the notable exception of the United States, where TEM- and SHV-type ESBLs have appeared to predominate. We have noted the emergence of ESBLs in our health care system (the University Health System in San Antonio, TX), especially in Escherichia coli isolates, that preferentially hydrolyze cefotaxime rather than ceftazidime, suggesting the possibility of CTX-M-type enzymes. Microbiology laboratory records were reviewed to identify ESBL-producing isolates and to compare the diameters of ceftazidime disk diffusion zones of inhibition to cefotaxime zone diameters. All isolates had been initially detected and confirmed using the procedures recommended by the Clinical and Laboratory Standards Institute. A total of 94 stored ESBL-producing isolates recovered between January 2000 and June 2006 (predominately from blood and normally sterile fluids) were retrieved for further study and screened using PCR primers specific for the presence of CTX-M, TEM, and SHV ESBLs. Only small numbers of retained ESBL-producing isolates were available for study in 2000 and 2002. The percentages of available ESBL-producing organisms in the following years were found to produce CTX-M enzymes: 2000, 25%; 2001, 10%; 2002, 0%; 2003, 60%; 2004, 69%; 2005, 89%; and 2006, 70%. The most common CTX-M-type ESBL was CTX-M-15, followed by CTX-M-16, CTX-M-8, and CTX-M-14. Comparing the disk diffusion zone diameters of cefotaxime and ceftazidime was helpful with the initial recognition of CTX-M-producing E. coli, which had an average cefotaxime zone diameter 7 mm smaller than the ceftazidime zone. However, comparing ceftazidime and cefotaxime zones for CTX-M-producing Klebsiella spp. was not helpful with initial recognition. CTX-M enzymes were also identified in Proteus mirabilis, Enterobacter spp., and Morganella morganii. Based on pulsed-field gel electrophoresis typing of the E. coli isolates, the CTX-M-producing isolates did not represent the spread of a single clone in the institution or in the community. In conclusion, CTX-M-type ESBLs are now the most common ESBL type isolated from patients in our health care system and may also be present but unrecognized in other U.S. locales.

Dataset Information

Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum ?-lactamases (ESBL).

Publications

Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).

Similar Datasets

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