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.

Comparison of Raman spectroscopy and two molecular diagnostic methods for Burkholderia cepacia complex species identification.

OBJECTIVES: Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa strains, colonize the respiratory tract of cyctic fibrosis patients. These strains are phenotypically difficult to discriminate, but differ greatly in their pathogenic potential and species identification is relevant. Here, three methods were compared for their diagnostic capacity. METHODS: A Bcc collection was analyzed with Raman spectroscopy, AFLP and rep-PCR analysis. RESULTS: Raman spectroscopy of 40 strains revealed high similarity. Rep-PCR and AFLP of respectively 96 and 112 strains revealed that Bcc strains could be distinguished from Pseudomonas strains. Both molecular methods allowed the identification of most Bcc species according to previous phenotypic and molecular characterization. CONCLUSION: Both AFLP and rep-PCR method data correspond with the previously reported species identification. However, Raman spectroscopy does not discriminate among P. aeruginosa and Bcc species and is therefore not useful as a diagnostic tool.

Molecular diagnostic analysis of outbreak scenarios.

In the current laboratory assignment, technical aspects of the polymerase chain reaction (PCR) are integrated in the context of six different bacterial outbreak scenarios. The "Enterobacterial Repetitive Intergenic Consensus Sequence" (ERIC) PCR was used to analyze different outbreak scenarios. First, groups of 2-4 students determined optimal ERIC-PCR conditions to validate the protocol and subsequently applied ERIC-PCR to identify genetic relatedness among bacterial strains. Based on these genetic fingerprints, students selected the outbreak cases from the patient samples and assessed the risk factors for the outbreak scenario. Finally, students presented their findings during a classroom presentation. The results indicated that the assignment successfully facilitated student learning on the technical aspects of (ERIC) PCR and clearly demonstrated the practical application of PCR in a clinical diagnostic setting. Additionally, the assignment was highly appreciated by the students.

Detection of colon flora in peritoneal drain fluid after colorectal surgery: can RT-PCR play a role in diagnosing anastomotic leakage?

A semi-quantitative Real-Time PCR strategy was developed to identify potential indicator organisms for anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture-negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture-negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method. The results showed that the semi-quantitative RT-PCR method has a clear potential to be useful as a powerful tool in early detection of anastomotic leakage.

A molecular blueprint of gene expression in hippocampal subregions CA1, CA3, and DG is conserved in the brain of the common marmoset.

Recent studies in rodents have shown that there are significant differences in gene expression profiles between the hippocampal subregions CA1, CA3, and DG. These differences in molecular make-up within the hippocampus most likely underlie the differences in morphology, physiology, and vulnerability to insults that exist between the subregions of the hippocampus and are as such part of the basic molecular architecture of the hippocampus. The aim of this study was to investigate at large scale whether these subregional differences in gene expression are conserved in the hippocampus of a nonhuman primate, the common marmoset. This study is very timely, given the recent development of the first marmoset-specific DNA microarray, exclusively containing sequences targeting transcripts derived from the marmoset hippocampus. Hippocampal subregions CA1, CA3, and DG were isolated by laser microdissection and RNA was isolated, amplified, and hybridized to the marmoset-specific microarray (EUMAMA) containing more than 1,500 transcripts expressed in the adult marmoset hippocampus. Large differences in expression were observed in particular between the DG region and both pyramidal subregions. Moreover, the subregion-specific patterns of gene expression showed a remarkable conservation with the rodent brain both in terms of individual genes and degree of differential expression. To our knowledge, this is the first study investigating large scale hippocampal gene expression in a nonhuman primate. The obtained expression profiles not only provide novel data on the expression of more than 1,500 transcripts per hippocampal subregion but also are of potential interest to neuroscientists interested in the role of the different subregions in learning and memory in the nonhuman primate brain.

Rapid glucocorticoid effects on the expression of hippocampal neurotransmission-related genes.

We previously assessed corticosterone mediated gene expression in acute explant hippocampal slices and found over 200 responsive genes 1, 3 and 5 h after glucocorticoid receptor (GR) activation by a brief corticosterone pulse. Interestingly, 1 h after GR activation all genes were downregulated, many of which are involved in hippocampal neurotransmission and plasticity. The aim of the current experiment was 1) to measure the expression of several of these neurotransmission-related genes that were corticosterone-responsive 1 h after GR-activation in an in vivo setting, 2) to elucidate in which hippocampal subregion these expression changes take place and 3) to assess the specificity of regulation by activated GRs. For this purpose, rats were subcutaneously injected with vehicle, corticosterone or corticosterone pretreated with GR-antagonist RU38486. One hour after the corticosterone injections, mRNA expression levels of 5 selected genes were measured using in situ hybridization. The mineralocorticoid receptor (MR), MAO-A, casein kinase 2 and voltage dependent potassium mRNA's, but not dynein mRNA, were rapidly downregulated in vivo after corticosterone administration in hippocampal subregions. Furthermore, RU38486 pretreatment reversed in all cases these effects, illustrating the GR-specificity of transcriptional regulation by corticosterone. The results are important for understanding the role of GR in pleiotropic control of hippocampal neurotransmission and plasticity, which is characterized by recovery of function transiently raised by excitatory input.

The dynamic pattern of glucocorticoid receptor-mediated transcriptional responses in neuronal PC12 cells.

The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid receptor (GR)-mediated transcriptional responses between different neuronal substrates and (ii) to assess the nature of these responses by differentiating between primary and downstream GR-responsive genes. For this purpose, nerve growth factor-differentiated catecholaminergic PC12 cells were used in which endogenous GRs were activated briefly with a high dose of corticosterone followed by gene expression profiling 1 and 3 h afterwards using Affymetrix GeneChips. The results revealed a strikingly similar temporal pattern to that which was reported previously in hippocampus, with only down-regulated genes 1 h after GR activation and the majority of genes up-regulated 3 h after GR activation. Real-time quantatitive PCR of transcripts in cycloheximide-treated cells showed that all five GR-responsive genes selected from the 1-h time point were primary responsive, whereas all four GR-responsive genes selected from the 3-h time point were downstream responsive. At the level of individual genes, the overlap with the previously generated hippocampal data sets was small, illustrating the cell-type specifity of GR-mediated genomic responses. Finally, we identified a number of interesting genes, such as SWI/SNF, synaptosomal-associated protein 25 and certain Rab proteins which may play a role in the effects of glucocorticoids on catecholaminergic neuronal functioning.

Acute activation of hippocampal glucocorticoid receptors results in different waves of gene expression throughout time.

Several aspects of hippocampal cell function are influenced by adrenal-secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)-induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR-mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR-activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down-regulated genes 1 h after GR-activation to both up and down regulated genes 3 h after GR-activation. After 5 h, the response was almost back to baseline. Additionally, real-time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR-responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid-mediated effects on hippocampal cell function.

Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression.

Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.