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Microarrays are powerful, highly parallel assays that are transforming microbiological diagnostics and research. The adaptation of microarray-based resequencing technology for microbial detection and characterization resulted in the development of a number assays that have unique advantages over other existing technologies. This technological platform seems to be especially useful for sensitive and high-resolution multiplexed diagnostics for clinical syndromes with similar symptoms, screening environmental samples for biothreat agents, as well as genotyping and whole-genome analysis of single pathogens.

A significant percentage of the human population is exposed to high levels of naturally occurring airborne dusts. Although the link between airborne particulate inhalation and a variety of respiratory diseases has long been established, little is known about the pathogenic role of the microbial component of the dust. In this study, we applied highly multiplexed PCR and a high-density resequencing microarray (RPM-TEI version 1.0) to screen samples of fine topsoil particles and airborne dust collected in 19 locations in Iraq and Kuwait for the presence of a broad range of human pathogens. The results indicated the presence of potential human pathogens, including Mycobacterium, Brucella, Coxiella burnetii, Clostridium perfringens, and Bacillus. The presence of Coxiella burnetii, a highly infectious potential biowarfare agent, was confirmed and detected in additional samples by use of a more sensitive technique (real-time PCR), indicating a high prevalence of this organism in the analyzed samples. The detection of potentially viable pathogens in breathable dusts from arid regions of Iraq and Kuwait underscores the importance of further study of these environments.

Microarray technology has revolutionized the detection and analysis of microbial pathogens. The success of this technology is evident from the various microarrays that have been developed for this purpose, variation in the density of probes, and the time ranges required for assay completion. Among these, high-density re-sequencing microarrays have demonstrated great potential for detecting bacterial, viral pathogens, and virulence markers. Resequencing microarrays use closely overlapping probe sets to determine a target organism's nucleotide sequence. Hybridization to a series of perfect matched probes provides confirmatory presence/absence information, while hybridization to mismatched probes reveals strain-specific single nucleotide polymorphism (SNP) data. This approach provides sequence information of the diagnostic regions of detected organisms that is considerably more informative over that provided from other microarray techniques.

Proanthocyanidins (PACs) are an abundant class of compounds found in a variety of plant materials. Here we demonstrate the application of these materials as capture molecules for the removal of bacterial lipopolysaccharide (LPS) from solution. PACs from whole cranberries, grape juice, black tea, and cranberry juice were purified and immobilized onto thiol-activated Sepharose beads. This material was used in pull-down type assays for the capture of LPS. The binding of LPS by PACs has been shown to compete with that of polymyxin B which is known to bind the lipid A component of LPS. Assays conducted in the presence of lipid A verified that at least some component of the LPS binding activity of the PACs is via the lipid A moiety. Molar comparison of polymyxin B to proanthocyanidins indicated that the Sepharose immobilized PACs have a binding affinity for LPS similar to that of polymyxin B.

Simultaneous testing for detection of infectious pathogens that cause similar symptoms (e.g., acute respiratory infections) is invaluable for patient treatment, outbreak prevention, and efficient use of antibiotic and antiviral agents. In addition, such testing may provide information regarding possible coinfections or induced secondary infections, such as virally induced bacterial infections. Furthermore, in many cases, detection of a pathogen requires more than genus/species-level resolution, since harmful agents (e.g., avian influenza virus) are grouped with other, relatively benign common agents, and for every pathogen, finer resolution is useful to allow tracking of the location and nature of mutations leading to strain variations. In this study, a previously developed resequencing microarray that has been demonstrated to have these capabilities was further developed to provide individual detection sensitivity ranging from 10(1) to 10(3) genomic copies for more than 26 respiratory pathogens while still retaining the ability to detect and differentiate between close genetic neighbors. In addition, the study demonstrated that this system allows unambiguous and reproducible sequence-based strain identification of the mixed pathogens. Successful proof-of-concept experiments using clinical specimens show that this approach is potentially very useful for both diagnostics and epidemic surveillance.

Rapid and accurate differential determination of viable versus nonviable microbes is critical for formulation of an appropriate response after pathogen detection. Sensors for rapid bacterial identification can be used for applications ranging from environmental monitoring and homeland defense to food process monitoring, but few provide viability information. This study combines the rapid screening capability of the array biosensor using an immunoassay format with methods for determination of viability. Additionally, cells captured by the immobilized antibodies can be cultured following fluorescence imaging to further confirm viability and for cell population expansion for further characterization, e.g., strain identification or antibiotic susceptibility testing. Finally, we demonstrate analysis of captured bacteria using the polymerase chain reaction (PCR). PCR results for waveguide-captured cells were 3 orders of magnitude more sensitive than the fluorescence immunoassay and can also provide additional genetic information on the captured microbes. These approaches can be used to rapidly detect and distinguish viable versus nonviable and pathogenic versus nonpathogenic captured organisms, provide culture materials for further analysis on a shorter time scale, and assess the efficacy of decontamination or sterilization procedures.

There is an increasing recognition that detailed nucleic acid sequence information will be useful and even required in the diagnosis, treatment and surveillance of many significant pathogens. Because generating detailed information about pathogens leads to significantly larger amounts of data, it is necessary to develop automated analysis methods to reduce analysis time and to standardize identification criteria. This is especially important for multiple pathogen assays designed to reduce assay time and costs. In this paper, we present a successful algorithm for detecting pathogens and reporting the maximum level of detail possible using multi-pathogen resequencing microarrays. The algorithm filters the sequence of base calls from the microarray and finds entries in genetic databases that most closely match. Taxonomic databases are then used to relate these entries to each other so that the microorganism can be identified. Although developed using a resequencing microarray, the approach is applicable to any assay method that produces base call sequence information. The success and continued development of this approach means that a non-expert can now perform unassisted analysis of the results obtained from partial sequence data.

Grooming is a proactive method to keep a ship’s hull free of fouling. This approach uses a frequent and gentle wiping of the hull surface to prevent the recruitment of fouling organisms. A study was designed to compare the community composition and the drag associated with biofilms formed on a groomed and ungroomed fouling release coating. The groomed biofilms were dominated by members of the Gammaproteobacteria and Alphaproteobacteria as well the diatomsNavicula,Gomphonemopsis,Cocconeis, andAmphora.Ungroomed biofilms were characterized by Phyllobacteriaceae, Xenococcaceae, Rhodobacteraceae, and the pennate diatomsCyclophora,Cocconeis, andAmphora.The drag forces associated with a groomed biofilm (0.75 ± 0.09 N) were significantly less than the ungroomed biofilm (1.09 ± 0.06 N). Knowledge gained from this study has helped the design of additional testing which will improve grooming tool design, minimizing the growth of biofilms and thus lowering the frictional drag forces associated with groomed surfaces. [ABSTRACT FROM AUTHOR]