Thomas F Ducey | PubMed Health

PubMed2006

A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.

Bacterial Proteins↗

PubMed2005

Identification of the iron-responsive genes of Neisseria gonorrhoeae by microarray analysis in defined medium.

To ensure survival, most bacteria must acquire iron, a resource that is sequestered by mammalian hosts. Pathogenic bacteria have therefore evolved intricate systems to sense iron limitation and regulate gene expression appropriately. We used a pan-Neisseria microarray to examine genes regulated in Neisseria gonorrhoeae in response to iron availability in defined medium. Overall, 203 genes varied in expression, 109 up-regulated and 94 down-regulated by iron deprivation. In iron-replete medium, genes essential to rapid bacterial growth were preferentially expressed, while iron transport functions, and predominantly genes of unknown function, were expressed in low-iron medium. Of those TonB-dependent proteins encoded in the FA1090 genome with unknown ligand specificity, expression of three was not controlled by iron availability, suggesting that these receptors may not be high-affinity transporters for iron-containing ligands. Approximately 30% of the operons regulated by iron appeared to be directly under control of Fur. Our data suggest a regulatory cascade where Fur indirectly controls gene expression by affecting the transcription of three secondary regulators. Our data also suggest that a second MerR-like regulator may be directly responding to iron availability and controlling transcription independent of the Fur protein. Comparison of our data with those recently published for Neisseria meningitidis revealed that only a small portion of genes were found to be similarly regulated in these closely related pathogens, while a large number of genes derepressed during iron starvation were unique to each organism.

Bacterial Proteins↗