Daniel D. Jones

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

Detection of Microbial Pathogens in Shellfish with Multiplex PCR

Abstract. Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55°C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelex™ 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was ≤101–102 cells following a “double” multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneComb™ (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneComb™ assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.

Expression of a foreign gene in Chlamydomonas reinhardtii

Genomic transformation of Chlamydomonas reinhardtii exposed to glass-bead abrasion was accomplished with a chimeric neomycin phosphotransferaseII (NPTII)-encoding gene (nos::npt) flanked by the nopaline synthase promoter and polyadenylation sequences obtained from the Ti plasmid of Agrobacterium tumefaciens. These sequences were in a plasmid (pGA482) which also contained gene nit1 encoding nitrate reductase of C. reinhardtii. Transformants were selected by their ability to grow on medium containing nitrate, and 52% of these was also resistant to kanamycin. Evidence for nos::npt expression includes: (1) hybridization with probes specific for npt, (2) demonstration of NPTII activity after electrophoresis of extracts, and (3) chromatographic identification of the reaction product of NPTII, kanamycin phosphate. The highly biased codon usage in Chlamydomonas does not preclude expression.

Conversion of cyanide to formate and ammonia by a pseudomonad obtained from industrial wastewater

SummaryA cyanide-degrading pseudomonad was isolated by selective enrichment in a chemostat inoculated with coke-plant activated sludge and maintained at a dilution rate of 0.042/h for 60 days with a feed of 10 mg/l cyanide. The isolate, a facultative methylotroph capable of growth on methanol and methylamine, degraded cyanide to formate and ammonia; it could utilize the released ammonia as a nitrogen source but did not further metabolize formate under the experimental conditions employed. Both cyanide-degrading enzyme activity and respiratory resistance to cyanide were inducible and were enhanced by repeated exposure to the compound. Cell-free extracts stoichiometrically converted cyanide to formate and ammonia in a reaction that did not require oxygen. Enzyme activity, lost upon dialysis, was restored by less than equimolar ratios of NAD(P)H or ascorbate to cyanide, indicating that the reductants did not function directly as co-enzymes.

Detection of viableVibrio cholerae by reverse-transcriptase polymerase chain reaction (RT-PCR)

The use of conventional PCR can amplify target DNA from both viable and nonviable cells ofVibrio cholera. Detection of only viable microbial pathogens in biological samples, especially clinical and food samples, is usually desired to ensure positive test results are associated with active agents, and not the remains of dead cells. Positive identifications caused by nonliving causative agents may lead to misguided decisions concerning the effectiveness of treatment, and whether patient treatment should be continued or whether the food should be discarded. Consequently, this work was directed toward development of a reverse-transcriptase polymerase chain reaction (RT-PCR)-based in vitro DNA amplification method, which specifically detects only viable cells. Total RNA from both viable and nonviable cells was purified by using a FastPrep Cell Disrupter (√Bio 101/Savant) and FastRNA extraction reagents (√Bio 101). The purified RNA was treated with DNase I (RNase-free) to avoid any amplification from the contaminating target DNA. An RT-PCR approach using this rapid and effective method for RNA purification showed amplification of the target mRNA only from the viable cells. The sensitivity of detection of viable cells ofV. cholerae was ≥103, which is well within the minimum number of cells (105–106) required for infection. The use of a reliable prokaryotic RNA extraction, method followed by RT-PCR amplification of the target mRNA can be used for specific detection of viable microbial pathogen, such asV. cholerae.

Biodegradabilities and Microbial Activities During Composting of Municipal Solid Waste In Bench-Scale Reactors

In the design of large scale, in-vessel systems, the biodegradability of the waste, the optimal nutritional conditions, and the length of time required to complete the high-rate phase are important. Because composting is a microbial process, the overall levels and activities of microorganisms influence the rate and extent of degradation. In this study, bench-scale reactors were used to determine the biodegradability of municipal solid waste, primarily office trash, and the effects of nutrient supplementation on biodegradability during six weeks of composting. Supplementation with nitrogen, phosphorus, vitamins, and trace minerals did not increase the biodegradability of the waste. Although the levels of specific microbial subpopulations varied, total numbers of microorganisms did not change significantly.

Microbial activities during composting of pulp and paper-mill primary solids

In this study, bench-scale aerobic reactors were used to monitor microbial activities and determine the degradability of pulp and paper-mill primary solids. Over the 43-day composting period, the level of microorganisms detected increased four-fold. However, based on amounts of CO2 produced, the increase expected was 19-fold, suggesting lysis of some cells in the population. The composition of the microbial population, determined through species identification by the BIOLOG® method, and by spot plating, changed throughout the composting process with Cellulomonas spp. not appearing until day 22. Chemical analyses suggested a fall of 33% in the cellulose content, but no other statistically significant chemical changes were observed.

Detection of Giardia in environmental waters by immuno-PCR amplification methods.

Abstract. Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR-amplified following “direct extraction” of Giardia DNA from seeded Cahaba river water concentrate with moderate turbidity (780 JTUs), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 × 105 JTUs) failed to amplify. However, if the cysts were first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex®100 treatment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 × 100 or 3 × 101 cysts/ml, respectively. If, however, the G. muris cysts used to seed even moderately turbid river waters (780 JTUs) were formalin treated (which is conventionally used for microscopic examination), neither direct extraction nor IMB purification methods yielded amplifiable DNA. Use of immunomagnetic beads to separate Giardia cysts from complex matrices of environmental surface waters followed by DNA release and PCR amplification of the target giardin gene improved the reliability of detection of this pathogen with the required sensitivity.

Putative anaerobic activity in aerated composts

It has been suggested that anaerobic microenvironments develop in aerobic composts, regardless of the aeration system used, and that anaerobic activity is responsible for odor generation and nitrogen losses. This study was designed to measure levels of microorganisms capable of anaerobic growth in two aerated composts: municipal solid waste, a relatively nutrient-rich compost, and pulp and paper-mill solid waste, which is relatively nutrient-poor. Anaerobic microorganisms were isolated from both composts at mesophilic and thermophilic temperatures. The majority of the anaerobic mesophiles were facultative anaerobes, whereas facultative, anaerobic thermophiles varied from 0 to 100%. Serially-diluted samples were spot-plated onto various media to preserve microbial consortia. Levels of aerobic and anaerobic exoenzyme production on spot-plates were similar on cell-wall, starch, and casein media. Although microbial levels on spread plates indicate that aerobes are present in much higher numbers than anaerobes (in 47 of 56 subsamples, 90% of the population were aerobes), microbial growth levels and exoenzyme production on spot-plates indicate that anaerobes may be responsible for a large portion (greater than or equal to 72%) of the metabolic activity in anaerobic microenvironments of aerobic composts.

Biodegradabilities and Microbial Activities During Composting of Oxidation Ditch Sludge

Best design of a compost system is dependent on knowing the degradability of the waste. Although many bulking agents used to adjust moisture and porosity consist of organic matter, it is usually assumed they contribute only a small proportion of the total degradable material in a compost mix. As the microorganisms degrade the organic compounds during composting, it is generally presumed that they grow, and increase in biomass and numbers. In this study, bench-scale compost reactors were used to determine the relative contributions of sewage sludge and the bulking agent (pine sawdust) to the biodegradability of a compost mix. Although the overall level of microorganisms did not change markedly during composting, subpopulations able to degrade selected macromolecules were present at varying levels. Populations capable of degrading bacterial cell walls and thereby of contributing to recycle of inorganic nutrients through biomass turnover were present throughout the composting period.