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Validation of virulence and epidemiology DNA microarray for identification and characterization of Staphylococcus aureus isolates.

ABSTRACT: The human pathogen Staphylococcus aureus is isolated and characterized using traditional culture and sensitivity methodologies that are slow and offer limited information on the organism. In contrast, DNA microarray technology can provide detailed, clinically relevant information on the isolate by detecting the presence or absence of a large number of virulence-associated genes simultaneously in a single assay. We have developed and validated a novel, cost-effective multiwell microarray for the identification and characterization of Staphylococcus aureus. The array comprises 84 gene targets, including species-specific, antibiotic resistance, toxin, and other virulence-associated genes, and is capable of examining 13 different isolates simultaneously, together with a reference control strain. Analysis of S. aureus isolates whose complete genome sequences have been determined (Mu50, N315, MW2, MRSA252, MSSA476) demonstrated that the array can reliably detect the combination of genes known to be present in these isolates. Characterization of a further 43 S. aureus isolates by the microarray and pulsed-field gel electrophoresis has demonstrated the ability of the array to differentiate between isolates representative of a spectrum of S. aureus types, including methicillin-susceptible, methicillin-resistant, community-acquired, and vancomycin-resistant S. aureus, and to simultaneously detect clinically relevant virulence determinants.

SUBMITTER: Spence RP

PROVIDER: S-EPMC2395118 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Validation of virulence and epidemiology DNA microarray for identification and characterization of Staphylococcus aureus isolates.

Spence Richard P RP Wright Victoria V Ala-Aldeen Dlawer A A DA Turner David P DP Wooldridge Karl G KG James Richard R

Journal of clinical microbiology 20080220 5

The human pathogen Staphylococcus aureus is isolated and characterized using traditional culture and sensitivity methodologies that are slow and offer limited information on the organism. In contrast, DNA microarray technology can provide detailed, clinically relevant information on the isolate by detecting the presence or absence of a large number of virulence-associated genes simultaneously in a single assay. We have developed and validated a novel, cost-effective multiwell microarray for the ...[more]

PMID: 18287310

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Project description:Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to approximately 128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

Project description:As genotyping of S. aureus is important for epidemiologic research and for hygiene management, methods are required for standardized fast and easily applicable evaluation of closely related epidemic strains with high prevalence in hospitals. In this single centre matched control study we compared a new commercially available DNA microarray (IdentiBAC) with standard spa-typing for S. aureus genotyping. Included in the study was a subgroup of 46 MRSA and matched 46 MSSA nasal isolates of the Saarland University Medical Center collected during a state-wide admission prevalence screening. Microarray (MA) and also spa-typing could easily differentiate the genetically diverse MSSA group. However, due to the predominance of CC5/t003 in the MRSA group a sufficient subtyping required analysis of more complex genetic profiles as was shown here by the MA comprising a total number of 334 different hybridization probes. The genetic repertoire of the MRSA group was characterized by more virulence genes as compared to the MSSA group. The standard evaluation of MA results by the original software into CCs, agr-, SCCmec- and capsule-types was substituted in the present study by implementation of multivariate subtyping of closely related CC5 isolates using three different bioinformatic methods (splits graph, cluster dendrogram, and principal component analysis). Each method used was applicable for standardized and highly discriminative subtyping with high concordance. We propose that the identified S. aureus subtypes with characteristic virulence gene profiles are presumably associated also with virulence and pathogenicity in vivo; however, this remains to be analyzed in future studies. MA was superior to spa-typing for epidemiologic and presumably also provide functional respectively virulence associated characterization of S. aureus isolates. This is of specific importance for the hospital setting. In future, MA could become a new standard test for S. aureus typing in combination with multivariate bioinformatic analysis.

Dataset Information

Validation of virulence and epidemiology DNA microarray for identification and characterization of Staphylococcus aureus isolates.

Publications

Validation of virulence and epidemiology DNA microarray for identification and characterization of Staphylococcus aureus isolates.

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