Timothy M. Straub

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

ABSTRACT We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei andDesulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacterand Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.

In vitro cell culture infectivity assay for human noroviruses

A 3-dimensional organoid human small intestinal epithelium model was used.

Towards a unified system for detecting waterborne pathogens

Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. This review discusses some of the challenges in developing a universal pathogen detection method, current technology that may be employed to overcome these challenges, and the remaining needs for developing an integrated pathogen detection and monitoring system for source or finished water.

Hazards from Pathogenic Microorganisms in Land-Disposed Sewage Sludge

Sewage sludge is a complex mixture of organic and inorganic compounds of biological and mineral origin that are precipitated from wastewater and sewage during primary, secondary, and tertiary sewage treatment. Present in these sludges are significant numbers of microorganisms that include viral, bacterial, protozoan, fungal, and helminth pathogens. The treatment of sludge to reduce biochemical oxygen demand, solids content, and odor is not always effective in reducing numbers of pathogens. This becomes a public health concern because the infectious dose for some of these pathogens may be as low as 1 particle (virus) to 50 organisms (Giardia). When sludge is applied to land for agricultural use and landfill compost, these pathogens can survive from days (bacteria) to months (viruses) to years (helminth eggs), depending on environmental conditions. Shallow aquifers can become contaminated with pathogens from sludge and, depending on groundwater flow, these organisms may travel significant distances from the disposal site. Communities that rely on groundwater for domestic use can become exposed to these pathogens, leading to a potential disease outbreak. Currently, methods to determine the risk of disease from pathogens in land-disposed sludge are inadequate because the sensitivity of pathogen detection is poor. The application of recombinant DNA technology (gene probes and polymerase chain reaction) to environmental samples may provide increased sensitivity for detecting specific pathogens in land-disposed sludge and greatly improved risk assessment models for our exposure to these sources of pathogens.

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays.

ABSTRACT Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.

Effect of anaerobic digestion on the occurrence of enteroviruses and Giardia cysts in sewage sludge

Abstract The disposal of anaerobically digested sewage sludge onto farmland has created the necessity to evaluate the fate of pathogens which are often present. The occurrence of enteroviruses and Giardia cysts in mesophilic anaerobically digested sludge before and after treatment, was monitored for a period of 14 months. The concentration of enteroviruses in sludge determined by Most Probable Number (MPN) ranged from 4.36 × 103 to 7.00 × 105MPN/Kg before anaerobic digestion and from 99.94%. The levels of intact Giardia cysts did not show any significant removal after sludge treatment. However, cyst viability could not be assessed by the method used for detection. Results of this...

Synergistic inactivation of Escherichia coli and MS-2 coliphage by chloramine and cupric chloride

Reaction of free chlorine with organic compounds in water during drinking water treatment may lead to the formation of potentially carcinogenic compounds known as trihalomethanes (THMs). Monochloramine and metals have been investigated as alternative disinfectants. However, the action of either disinfectant alone requires greater concentrations and longer contact times compared to free chlorine. This experiment evaluated the efficacy of combining monochloramine (1–2.5 mg/l) and copper in the form of cupric chloride (0.1–0.8 mg/l) to determine if inactivation of MS-2 coliphage and Escherichia coli was synergistic and thereby decreasing the concentration and contact time for adequate inactivation of these organisms. Combination of 5 mg/l monochloramine and 0.1–0.4 mg/l cupric chloride was sufficient to produce a 3 log10 inactivation of MS-2 coliphage after 10 min. Nearly 120 min was required for the same log10 inactivation of MS-2 using 5 mg/l monochloramine alone and less than a 0.5 log10 reduction was observed after 120 min using 0.4 mg/l cupric chloride alone. A 6 log10 reduction of E. coli was observed after 10- and 20-min exposures to 2.5 mg/l monochloramine and 0.8 or 0.4 mg/l cupric chloride, respectively. To achieve the same inactivation of E. coli using monochloramine alone, a concentration and contact time of 5 mg/l for 60 min was required. No inactivation of E. coli was observed after exposure to 0.4 or 0.8 mg/l cupric chloride after 60 min. Synergism was demonstrated in the inactivation of both organisms using the combined chloramine copper system.

Removal of PCR inhibiting substances in sewage sludge amended soil

Current methods for the detection of enteric viruses in soil involve elution of viruses from soil colloids using beef extract or other proteins. These eluates are then assayed in cell culture and observed daily for cytopathic effects (CPE). While this method is suitable for detection of enteric viruses by cell culture, these eluates contain humic acids and heavy metals that interfere with polymerase chain reaction (PCR) detection. Using beef extract eluates prepared from sludge amended soil, 10 different methods of eluate purification were evaluated for their ability to remove PCR inhibition and maximize sensitivity. The treatment method providing the greatest sensitivity of poliovirus detection by PCR was the combination of Sephadex G-50 and Chelex-100. Using this method 2 plaque forming units (PFU) could be detected after reverse transcription and 30 cycles of PCR. Thirty (30) cycles of seminested PCR were performed on these samples to verify nucleic acid sequences and increase sensitivity after the first 30 cycles of PCR. Using seminested PCR, sensitivity of detection using the Sephadex G-50 and Chelex-100 treatment method to 0.2 PFU. In addition to providing excellent sensitivity for viruses in sludge amended soils, this treatment method is relatively simple compared to other methods.

Estimated copy number of Bacillus anthracis plasmids pXO1 and pXO2 using digital PCR

We evaluated digital PCR (dPCR) to directly enumerate plasmid and chromosome copies in three strains of Bacillus anthracis. Copy number estimates based on conventional quantitative PCR (qPCR) highlighted the variability of using qPCR to measure copy number whereas estimates based on direct sequencing are comparable to dPCR.

Use of a Novel Fluidics Microbead Trap/Flow-Cell Enhances Speed and Sensitivity of Bead-Based Bioassays

Automated devices and methods for biological sample preparation often use surface functionalized microbeads (superparamagnetic or nonmagnetic) to allow capture, purification, and preconcentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or nonmagnetic functionalized beads that allow samples and reagents to be efficiently perfused over a microcolumn of beads. This approach yields enhanced mass transport and up to fivefold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summary results are given that highlight the analytical performance improvements obtained for automated microbead processing systems using novel microbead trap/flow-cells for various applications including (1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; (2) capture of nucleic acids using oligonucleotide-coupled polystyrene beads with flow cytometry detection; and (3) capture of Escherichia coli 0157:H7 from 50-mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.