Unknown,Transcriptomics,Genomics,Proteomics

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Detection of virulence and antibiotic resistance gene families

ABSTRACT: Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known and novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. Keywords: detection, pathogen, virulence mechanism In this report, we describe the process used to design our first generation functional array for highly sensitive detection of virulence and antibiotic resistance gene families. We discuss the probe design algorithms, including virulence gene sequence selection, and our protocols for sample preparation, amplification, labeling, hybridization, and data analysis. We present the results from experiments designed to assess whether the array can detect virulence gene orthologs from organisms without perfect match probes on the array, using both targeted mismatch probes and hybridizations to DNA from other organisms. Also, we report the results from limit of detection studies, using known amounts of bacterial DNA spiked into aerosol samples to measure the minimal concentration required for detection of virulence elements against a complex background.

ORGANISM(S): Staphylococcus aureus subsp. aureus Mu50

SUBMITTER: Crystal Jaing

PROVIDER: E-GEOD-11010 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A functional gene array for detection of bacterial virulence elements.

Jaing Crystal C Gardner Shea S McLoughlin Kevin K Mulakken Nisha N Alegria-Hartman Michelle M Banda Phillip P Williams Peter P Gu Pauline P Wagner Mark M Manohar Chitra C Slezak Tom T

PloS one 20080514 5

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistan ...[more]

PMID: 18478124

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Project description:The opportunistic pathogen, Staphylococcus aureus, encounters a wide variety of fluid shear levels within the human host, which may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. Using rotating-wall vessel bioreactors to create a physiologically-relevant, low fluid shear environment, S. aureus was evaluated for cellular responses that could impact its colonization and virulence. S. aureus cells grown in a low fluid shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed the down-regulation of the RNA chaperone Hfq, which parallels low fluid shear responses of certain Gram negative organisms. This is the first study to report an Hfq association to fluid shear in a Gram positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggest S. aureus responds to a low fluid shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during initial host pathogen interaction. Genetic expression profiles of Staphylococcus aureus cultured under low fluid shear conditions was compared to control cultures of S. aureus which was cultured in identical hardware in an orientation disrupting the low fluid shear effect. Samples from the same date of culture were compared (control 21:low 21 and control 30: low 30). S. aureus was cultured for 20 hours in either the low fluid shear or control orientated rotating wall vessel (RWV) bioreactor at which point the cells were removed and RNA extracted. At 20 hours, both cultures were in the same stage of growth (stationary phase) and at this point phenotypic differences between control and low fluid shear cultures were noted.

Dataset Information

Detection of virulence and antibiotic resistance gene families

Publications

A functional gene array for detection of bacterial virulence elements.

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