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Biomedical subjects

Daniel Lew

Publications and source records attributed to Daniel Lew.

At least 19 recordsLinked to original sources
PubMed2006

Exploring glycopeptide-resistance in Staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers.

BACKGROUND: To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. RESULTS: In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. CONCLUSION: Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

Anti-Bacterial Agents↗
PubMed2006

Use of oligoarrays for characterization of community-onset methicillin-resistant Staphylococcus aureus.

Until recently, methicillin-resistant Staphylococcus aureus (MRSA) was considered the prototype of a hospital-acquired bacterial pathogen. However, recent reports have shown that MRSA has now emerged in the community. Characterization of specific markers for distinguishing the origin of isolates could contribute to improved knowledge of MRSA epidemiology. The release of whole-genome sequences of hospital- and community-acquired S. aureus strains allowed the development of whole-genome content analysis techniques, including microarrays. We developed a microarray composed of 8,191 open reading frame-specific oligonucleotides covering >99% of the four sequenced S. aureus genomes (N315, Mu50, MW2, and COL) to evaluate gene contents of hospital- and community-onset S. aureus strains. In parallel, pulsed-field gel electrophoresis, variable number of tandem repeats, antibiogram, staphylococcal cassette chromosome-mec element typing, and presence of the Panton-Valentine leukocidin gene were evaluated in a collection of 15 clinical isolates. Clusters obtained with microarrays showed a high degree of similarity with those obtained by pulsed-field gel electrophoresis or variable number of tandem repeats. Clusters clearly segregated hospital-onset strains from community-onset strains. Moreover, the microarray approach allowed definition of novel marker genes and chromosomal regions specific for given groups of isolates, thus providing better discrimination and additional information compared to pulsed-field gel electrophoresis and variable number of tandem repeats. Finally, the comparative genome hybridization approach unraveled the occurrence of multiple horizontal transfer events leading to community-onset MRSA as well as the need for a specific genetic background in recipient strains for both the acquisition and the stability of the mec element.

Adult↗
PubMed2006

Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.

Bacterial Proteins↗
PubMed2005

Tracking methicillin-resistant Staphylococcus aureus clones in Colombian hospitals over 7 years (1996-2003): emergence of a new dominant clone.

Worldwide dissemination of methicillin-resistant Staphylococcus aureus (MRSA) clones is a well-characterised phenomenon. Two hundred isolates of MRSA recovered from 17 Colombian hospitals collected between 2001 and 2003 were characterised by pulsed-field gel electrophoresis (PFGE). A new dominant electrophoretic pattern unrelated to previously characterised clones in Colombia was detected in 137 (68.5%) of these isolates. Only 40 (20%) isolates still showed a pattern closely related to a previously described dominant clone. The new electrophoretic pattern was indistinguishable from a cluster of isolates recovered in Chile between 1996 and 1998. Isolates from this clonal cluster exhibited multidrug resistance but were susceptible to linezolid and glycopeptides. The results indicate a shift in the population genetics of Colombian MRSA and confirm dissemination of the Chilean clone for the first time.

Anti-Bacterial Agents↗
PubMed2005

Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription.

Bacteremia↗
PubMed2005

Evidence of an intracellular reservoir in the nasal mucosa of patients with recurrent Staphylococcus aureus rhinosinusitis.

Severe infections due to Staphylococcus aureus require prolonged therapy for cure, and relapse may occur even years after the first episode. Persistence of S. aureus may be explained, in part, by nasal carriage of S. aureus, which occurs in a large percentage of healthy humans and represents a major source of systemic infection. However, the persistence of internalized S. aureus within mucosal cells has not been evaluated in humans. Here, we provide the first in vivo evidence of intracellular reservoirs of S. aureus in humans, which were assessed in endonasal mucosa specimens from patients suffering from recurrent S. aureus rhinosinusitis due to unique, patient-specific bacterial clonotypes. Heavily infected foci of intracellular bacteria located in nasal epithelium, glandular, and myofibroblastic cells were revealed by inverted confocal laser scan fluorescence and electron microscopic examination of posttherapy intranasal biopsy specimens from symptom-free patients undergoing surgery on the sinuses. Intracellular residence may provide a sanctuary for pathogenic bacteria by protecting them from host defense mechanisms and antibiotic treatment during acute, recurrent S. aureus rhinosinusitis.

Biopsy↗
PubMed2005

High-density DNA probe arrays for identification of staphylococci to the species level.

Rapid and accurate identification and speciation of staphylococci clinical isolates is important for predicting medical pathology. We evaluated the ability of a high-density DNA probe array based on 16S rDNA sequences to identify Staphylococcus species. Correct identification was observed for 185 out of the 201 strains (92%). Of the 33 tested species, the array was able to correctly identify 30 of them. The total time required for identification of 4 isolates was 5 h. Such a tool represents a powerful method for routine microbiological diagnostic as well as for epidemiological studies.

Animals↗
PubMed2005

The inhibition of neutrophil antibacterial activity by ultra-high molecular weight polyethylene particles.

Following infection, bacterial killing by polymorphonuclear leukocytes (neutrophils) is the main host defense against bacteria. Our hypothesis is that particles of ultra-high molecular weight polyethylene (UHMWP) may impair local neutrophil function and consequently reduce neutrophil bacterial killing. To determine how the in vitro phagocytic-bactericidal activity of neutrophils was affected by exposure to wear particles, tests were run comparing the effects of different particle composition, and different concentrations and sizes of UHMWP particles. There was a significant correlation between the number of particles and the decrease in neutrophil bactericidal activity (p<0.01), and the greatest effect was obtained with a concentration of 10(7) UHMWP/ml. There was a significant decrease in neutrophil bactericidal activity by incubation with particles of 0.1-5 microm (p<0.01), but not with larger size. The results suggest that neutrophil functional defects triggered by the presence of UHMWP particles may potentially contribute to the susceptibility of loose implants to bacterial infections.

Biocompatible Materials↗
PubMed2005

Evaluation of a high-density oligonucleotide array for characterization of grlA, grlB, gyrA and gyrB mutations in fluoroquinolone resistant Staphylococcus aureus isolates.

Using high-density oligonucleotide array technology, 30 Staphylococcus aureus strains were studied for the presence of mutations in genes involved in fluoroquinolone resistance: grlA, gyrA, grlB and gyrB. For the two most important genes, gyrA and grlA, correlation with sequencing reached 95.1%. If all genes were considered, correlation was 88.8%.

Codon↗
PubMed2005

Fusarium peritonitis concomitant to kidney transplantation successfully managed with voriconazole: case report and review of the literature.

Fusarium infections in solid organ transplant recipients are often localized, occur later in the post-transplantation period, and have a better outcome than fusarial infections in patients with hematologic malignancies or bone marrow transplants. We report the first case of proven peritonitis caused by Fusarium species in a renal transplant recipient which is also the first successfully managed with voriconazole. We also review previously reported cases of fusarial infection in solid organ transplant recipients.

Antifungal Agents↗
PubMed2005

Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates.

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.

Automation↗
PubMed2005

Molecular identification of Fusarium species in onychomycoses.

BACKGROUND: Fusarium species are isolated from about 3% of onychomycoses in the Swiss native population. On the basis of macroscopic characters and microscopic examination of the cultures, identification of Fusarium often remains difficult or uncertain because of variations from one isolate to another and overlapping characteristics between species. OBJECTIVE: To obtain information about the prevailing species of Fusarium collected from onychomycoses. METHODS: An analysis of the Fusarium specimens isolated in the Department of Dermatology at the University Hospital of Lausanne was conducted during a 2-year period (71 isolates). A 311-bp fragment of the gene encoding 28S rDNA was amplified by PCR and sequenced. DNA sequences were compared to those available for reference strains. RESULTS: Fusarium oxysporum was the most frequently isolated species, accounting for 54% of the isolates. F. proliferatum and 4 taxons belonging to the F. solani species complex were identified with an appreciable frequency ranging from 4 to 14%. CONCLUSION: The Fusarium species identified were the same as those known to cause disseminated fusariosis in immunocompromised patients. The presence of these Fusarium species in onychomycoses warrants that careful attention should be paid to abnormal nails before beginning immunosuppressive treatments in patients.

Base Sequence↗
PubMed2004

Impact of enteral versus parenteral nutrition on the incidence of fungal infections: a retrospective study in ICU patients on mechanical ventilation with selective digestive decontamination.

BACKGROUND: Infections are an important cause of morbidity and mortality in patients in intensive care units (ICUs). Fungal infections have increased substantially over recent years and fungi have become one of the important pathogens in ICU patients. This study tests the hypothesis that the incidence of fungal infections is lower in critically ill patients under mechanical ventilation receiving enteral rather than total parenteral nutrition. METHODS: By using a prospectively built database, we analyzed retrospectively the charts of 110 critically ill, intubated patients hospitalized in surgical and medical ICUs and receiving selective digestive topical decontamination (SDD), which consisted of administering non-absorbable antibiotics. Patients without contraindications, and expected to be intubated for more than 72 h, received enteral nutrition within 24 h after intubation. Patients with contraindications for enteral nutrition received total parenteral nutrition, which was discontinued when the criteria for enteral nutrition were met. The incidence of fungal infections in both subgroups of patients was compared. RESULTS: Seventy-nine patients received enteral nutrition and 31 total parenteral (10 patients did not meet the inclusion criteria). Both subgroups were similar with regard to their APACHE II score, age, sex distribution and comorbidities at the time of study entry. No difference was observed in the rate of fungal infections between enteral nutrition (5/29) and total parenteral nutrition (7/71) patient groups. CONCLUSION: No significant decrease in the incidence of fungal infections in critically ill patients receiving SDD was observed between those receiving enteral and total parenteral nutrition.

Adolescent↗
PubMed2004

The value of bacterial culture during clean orthopedic surgery: a prospective study of 1,036 patients.

OBJECTIVE: To determine whether bacterial cultures of the wounds of patients undergoing clean orthopedic surgery would help predict infection. METHODS: During 1 year, 1,256 cultures were performed for 1,102 patients who underwent clean orthopedic surgery. Results were analyzed to evaluate their ability to predict postoperative infection. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value of the cultures were 38%, 92%, 7%, and 99%, respectively. CONCLUSIONS: Cultures performed during clean orthopedic surgery were not useful for predicting postoperative infection.

Adolescent↗