Search

The infections caused by extended-spectrum β-lactamase (ESBL)-producing organisms are associated with increased mortality. The real-time polymerase chain reaction (PCR) method, which enables detection of ESBLs directly from patients' clinical material, was developed. This study focused on blaCTX-M and blaSHV determination in endotracheal aspirates. Each sample was identified with standard microbiological procedures and simultaneously analyzed for the presence of nucleic acids, which encode CTX-M and SHV ESBL enzymes using real-time PCR. A total of 341 samples were investigated. In the set, 27 ESBL-positive samples were identified by phenotypic methods, while 60 positive samples were identified by the PCR method. Of the 60 PCR-positive samples, 58 were positive for the blaCTX-M. In two samples, the ESBL blaSHV-ESBL gene was detected. One phenotypically positive sample was PCR negative. The real-time PCR assay does not require a cultivation step and therefore enables detection of ESBL in 6 hours. The rapid method is necessary for early and adequate antimicrobial treatment.

Serious bloodstream infections are a significant complication in critically ill patients. The treatment of these infections has become more difficult because of the increasing prevalence of multiresistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA). Rapid differentiation of low number of MRSA from methicillin-susceptible S. aureus (MSSA) cells (10(1)-10(2) cells mL(-1)) in blood is necessary for fast effective antibiotic therapy. Currently, three groups of techniques, phenotyping, genotyping, and mass spectrometry, are used for MRSA and MSSA strains differentiation. Most of these techniques are time-consuming. PCR and other molecular techniques allow the detection and differentiation between MSSA and MRSA directly from blood cultures. These methods alone are rapid and they have good reproducibility and repeatability. Potential disadvantages of the genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. Recently, capillary electrophoresis (CZE) was successfully used to differentiate between the agar-cultivated MRSA and MSSA strains in fused silica capillaries etched with supercritical water and modified with (3-glycidyloxypropyl)trimethoxysilane. The possible use of CZE as a fast and low-cost method for distinguishing between the blood-incubated MRSA or MSSA cells has been tested in this manuscript. Our goal was to test low amounts of bacteria (∼10(2) cell mL(-1)) similar to those in clinical samples. The migration times of the purified blood-incubated cells and the agar-cultivated cells were different from each other. However, their isoelectric point was the same for all strains., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Identification and prevention of Staphylococcus aureus-caused infections may benefit from a fast and dependable method to distinguish between the methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. The current methods involving polymerase chain reaction and/or other molecular tests are usually laborious and time-consuming. We describe here a fast and low-cost method employing capillary zone electrophoresis (CZE) to distinguish between MRSA and MSSA. The method makes use of a supercritical water-treated fused silica capillary, the inner surface of which has subsequently been modified with (3-glycidyloxypropyl)trimethoxysilane. With optimized proportions of suitable additives to the background electrolyte, a CZE separation of MRSA from MSSA may be completed within 12 min. The cells were baseline-resolved, and resolution was determined to be 3.61. The isoelectric points of MSSA and MRSA were found to be the same for both groups of these strains, pI = 3.4.

Objectives: A new method has been developed for detecting genes determining the extended-spectrum beta-lactamase (ESBL) phenotype directly from patients' clinical material. The method enables detection of the bla(CTX-M) gene encoding CTX-M beta-lactamases and the bla(SHV) gene variants with real-time PCR technology using locked nucleic acid oligonucleotides., Material and Methods: In this pilot study, tracheal aspirates obtained from patients with mechanical ventilation hospitalized at Department of Anaesthesiology and Resuscitation of the University Hospital in Olomouc between 1st March and 30th December 2010 period were tested. Each sample was identified with standard microbiological procedures including phenotypic determination of ESBL-positive enterobacteria. At the same time, each sample was analyzed for the presence of nucleic acids (DNA) which encode CTX-M and SHV ESBL using real-time PCR., Results: 150 samples of tracheal aspirates from 71 patients were included into testing. In the set, 13 (8.7%) ESBL-positive samples were identified by culture methods while 27 (18 %) positive samples were identified by the real-time PCR method. Of the 27 PCR-positive samples, 24 were positive for the bla(CTX) gene; in 2 samples, the ESBL bla(SHV) gene was detected, and both genes were present in 1 sample. All culture-positive samples were also PCR-positive for the presence of bla(CTX) and/or bla(SHV) sequences., Conclusions: The new real-time PCR assay is likely to shorten the time for detection of enterobacteria producing SHV and CTX-M beta-lactamases from 48 to 6 hours. It enables ESBL-positive enterobacteria determination in tracheal aspirates of patients suffered from life-threatening nosocomial pneumonia where the early introduction of adequate antimicrobial treatment plays the important role.