Antimicrobial susceptibility testing of Gram-positive and -negative bacterial isolates directly from spiked blood culture media with Raman spectroscopy.

Patients suffering from bacterial bloodstream infections have an increased risk of developing systematic inflammatory response syndrome (SIRS), which can result in rapid deterioration of the patients' health. Diagnostic methods for bacterial identification and antimicrobial susceptibility tests are time-consuming. The aim of this study was to investigate whether Raman spectroscopy would be able to rapidly provide an antimicrobial susceptibility profile from bacteria isolated directly from positive blood cultures. First, bacterial strains (n = 133) were inoculated in tryptic soy broth and incubated in the presence or absence of antibiotics for 5 h. Antimicrobial susceptibility profiles were analyzed by Raman spectroscopy. Subsequently, a selection of strains was isolated from blood cultures and analyzed similarly. VITEK®2 technology and broth dilution were used as the reference methods. Raman spectra from 67 antibiotic-susceptible strains showed discriminatory spectra in the absence or at low concentrations of antibiotics as compared to high antibiotic concentrations. For 66 antibiotic-resistant strains, no antimicrobial effect was observed on the bacterial Raman spectra. Full concordance with VITEK®2 data and broth dilution was obtained for the antibiotic-susceptible strains, 68 % and 98 %, respectively, for the resistant strains. Discriminative antimicrobial susceptibility testing (AST) profiles were obtained for all bacterial strains isolated from blood cultures, resulting in full concordance with the VITEK®2 data. It can be concluded that Raman spectroscopy is able to detect the antimicrobial susceptibility of bacterial species isolated from a positive blood culture bottle within 5 h. Although Raman spectroscopy is cheap and rapid, further optimization is required, to fulfill a great promise for future AST profiling technology development.

Good performance of the SpectraCellRA system for typing of methicillin-resistant Staphylococcus aureus isolates.

Typing of methicillin-resistant Staphylococcus aureus (MRSA) remains necessary in order to assess whether transmission of MRSA occurred and to what extent infection prevention measures need to be taken. Raman spectroscopy (SpectraCellRA [SCRA]; RiverD International, Rotterdam, The Netherlands) is a recently developed tool for bacterial typing. In this study, the performance (typeability, discriminatory power, reproducibility, workflow, and costs) of the SCRA system was evaluated for typing of MRSA strains isolated from patients and patients' household members who were infected with or colonized by MRSA. We analyzed a well-documented collection of 113 MRSA strains collected from 54 households. The epidemiological relationship between the MRSA strains within one household was used as the gold standard. Pulsed-field gel electrophoresis (PFGE) was used for discrepancy analysis. The results of SCRA analysis on the strain level corresponded with epidemiological data for 108 of 113 strains, a concordance of 95.6%. When analyzed at the household level, the results of SCRA were correct for 49 out of 54 households, a concordance of 90.7%. Concordance on the strain level with epidemiological data for PFGE was 93.6% (103/110 isolates typed). Concordance on the household level with epidemiological data for PFGE was 93.5% (49/53 households analyzed). With PFGE regarded as the reference standard, the conclusions reached with Raman spectroscopy were identical to those reached with PFGE in 100 of 105 cases (95.2%). The reproducibility of SCRA was found to be 100%. We conclude that the SpectraCellRA system is a fast, easy-to-use, and highly reproducible typing platform for outbreak analysis that can compete with the currently used typing techniques.

In vitro evaluation of the performance of Granada selective enrichment broth for the detection of group B streptococcal colonization.

A broth for the screening of group B streptococcal (GBS) carriage during pregnancy is about to be introduced. Simulating conditions in everyday practice, we have compared the sensitivity of this Granada tube broth (GT) with that of classical Amies transport medium (AT) in vitro. A total of 1,485 GT and 1,485 AT were tested with 33 well-characterized GBS strains in three different concentrations, five different incubation times, and three different temperatures. After initial incubation at room temperature (RT) or 4°C, GT were placed at 37°C. GT were scored for the presence of orange pigment. GT and AT were subcultured on blood agar (BA). Pigment was observed in 98% of GT incubated at 37°C. GBS could be cultured in 91%, 73%, and 55% of GT incubated at 37°C, RT, or 4°C, respectively. For AT, these percentages were only 20% at 37°C, 52% at RT, and 59% at 4°C. When GT initially incubated at RT or 4°C were subsequently incubated at 37°C, the sensitivity improved significantly. We conclude that GT is a more sensitive GBS transport and culture medium than the conventional method, especially for low inocula and prolonged transport/incubation times. GT does not exclude the presence of GBS, and should always be incubated at 37°C and subcultured on solid agar for optimal sensitivity.

External quality assessment of the molecular diagnostics and genotyping of meticillin-resistant Staphylococcus aureus.

Two multicentre external quality assessments (EQA) for the molecular detection and genotyping of meticillin-resistant Staphylococcus aureus (MRSA) were arranged. Firstly, 11 samples containing various amounts of inactivated MRSA strains, meticillin-susceptible S. aureus (MSSA), meticillin-resistant coagulase-negative staphylococci (MRCoNS) or Escherichia coli were distributed to 82 laboratories. Samples containing 102 or 103 MRSA cells were correctly scored in only 16 and 46% of the datasets returned, respectively. Two of the used MSSA strains contained an SCCmec cassette lacking the mecA gene. There was a marked difference in the percentage of correct results for these two MSSA strains (37 and 39%) compared to the MSSA strain lacking the SCCmec cassette (88%). Secondly, a panel for MRSA genotyping, consisting of ten samples (two identical, three genetically related and five unique strains) was distributed to 19 laboratories. Seventy-three percent of the datasets recorded all samples correctly. Most pulsed-field gel electrophoresis (PFGE) protocols proved to be suboptimal, resulting in inferior resolution in the higher or lower fragment regions. The performance of molecular diagnostics for MRSA shows no significant changes since our first EQA in 2006. The first molecular typing results are encouraging. Both assessments indicate that programme expansion is required and that major performance discrepancies continue to exist.

Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens.

The performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference method. A total of 824 respiratory specimens, comprised of sputa, bronchoalveolar lavage fluid, and bronchial and tracheal aspirates from 580 patients, were evaluated. Out of 824 clinical specimens, 109 specimens from 43 patients were culture positive for M. tuberculosis. Of these 109 specimens, 67 were smear positive, 85 were positive by the COBAS AMPLICOR MTB test, and 94 were positive by the BDProbeTec ET. Of the 715 culture-negative specimens, 17 were positive by the auramine staining, 11 were positive by the COBAS AMPLICOR MTB test, and 12 were positive by the BDProbeTec ET. After discrepancy analysis and review of the patients' clinical data, 130 specimens from 50 patients were considered "true-positive" specimens. This resulted in the following sensitivities: microscopy, 61.5%; COBAS AMPLICOR MTB test, 78.0%; and BDProbeTec ET, 86.2%. The specificities of each system, based on the clinical diagnosis, were 99.7% for microscopy, 99.9% for the COBAS AMPLICOR MTB test, and 99.9% for the BDProbeTec ET. The data presented represent a considerable number of specimens evaluated with a considerable number of culture- and auramine-positive and culture-positive and auramine-negative results and therefore give a realistic view of how the data should be interpreted in a daily routine situation. Specifically, the data with regard to the culture-positive and auramine-negative specimens are useful, because in a routine situation, auramine-negative specimens are sometimes accepted, on clinical indications, to be analyzed by an amplification method.

The prevalence and clonal expansion of high-level gentamicin-resistant enterococci isolated from blood cultures in a Dutch university hospital.

We studied the prevalence and clonality of high-level gentamicin-resistant enterococci (HLGRE) in a Dutch university hospital. Of 238 enterococcal strains isolated from blood cultures between 1991 and 1997, 57 were HLGRE. Genomic analysis of these strains revealed 19 different genotypes, two of which were encountered more frequently [type A (12/57), type B (23/57)]. The spread of these types largely explained the rise in HLGRE incidence from 14% in 1991 to 31% in 1997. However, the contribution of unique strains to the total HLGRE burden also increased from 4% to 16%. We conclude that both clonal expansion and the emergence of unique HLGRE have contributed significantly to the increasing incidence of HLGRE.