A species independent universal bio-detection microarray for pathogen forensics and phylogenetic classification of unknown microorganisms

<p>Abstract</p> <p>Background</p> <p>The ability to differentiate a bioterrorist attack or an accidental release of a research pathogen from a naturally occurring pandemic or disease event is crucial to the safety and security of this nation by enabling an appropriate and rapid response. It is critical in samples from an infected patient, the environment, or a laboratory to quickly and accurately identify the precise pathogen including natural or engineered variants and to classify new pathogens in relation to those that are known. Current approaches for pathogen detection rely on prior genomic sequence information. Given the enormous spectrum of genetic possibilities, a field deployable, robust technology, such as a universal (any species) microarray has near-term potential to address these needs.</p> <p>Results</p> <p>A new and comprehensive sequence-independent array (Universal Bio-Signature Detection Array) was designed with approximately 373,000 probes. The main feature of this array is that the probes are computationally derived and sequence independent. There is one probe for each possible 9-mer sequence, thus 4⁹(262,144) probes. Each genome hybridized on this array has a unique pattern of signal intensities corresponding to each of these probes. These signal intensities were used to generate an un-biased cluster analysis of signal intensity hybridization patterns that can easily distinguish species into accepted and known phylogenomic relationships. Within limits, the array is highly sensitive and is able to detect synthetically mixed pathogens. Examples of unique hybridization signal intensity patterns are presented for different <it>Brucella </it>species as well as relevant host species and other pathogens. These results demonstrate the utility of the UBDA array as a diagnostic tool in pathogen forensics.</p> <p>Conclusions</p> <p>This pathogen detection system is fast, accurate and can be applied to any species. Hybridization patterns are unique to a specific genome and these can be used to decipher the identity of a mixed pathogen sample and can separate hosts and pathogens into their respective phylogenomic relationships. This technology can also differentiate between different species and classify genomes into their known clades. The development of this technology will result in the creation of an integrated biomarker-specific bio-signature, multiple select agent specific detection system.</p

Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates

ABSTRACTBackground Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres’ respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case.Results Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains.Conclusions We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts

High-level Relatedness among Mycobacterium abscessus subsp. massiliense Strains from Widely Separated Outbreaks

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol. Download MP3  Length: 1:2

Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes

We report the genome sequence of Theileria parva, an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum, and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.4 page(s

Genome sequence of the human malaria parasite Plasmodium falciparum

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria