A comparison of Roche Kinetic Interaction of Microparticles in Solution (KIMS) assay for cannabinoids and GC-MS analysis for 11-nor-9-carboxy-delta9-tetrahydrocannabinol.

In this study, we investigated the effectiveness of the Roche Kinetic Interaction of Microparticles in Solution (KIMS) screening assay for cannabinoid metabolites. Urine specimens (N = 1689) were collected during elimination of cannabinoids from 25 subjects with a history of marijuana use. Specimens were analyzed concurrently for cannabinoid metabolites by a customized Department of Defense (DOD) cannabinoid KIMS kit (50-ng/mL cutoff) and for 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THC-COOH) by GC-MS (15-ng/mL cutoff). As compared to GC-MS results, the sensitivity, specificity, and efficiency of the KIMS assay were 69.7%, 99.8%, and 88.6%, respectively. Many of the false-negative results had GC-MS concentrations between 15 and 26 ng/mL (N = 151). The cannabinoid screening results for the DOD samples tested by the laboratory during the same 8-month period were also evaluated. The linear regression analyses of GC-MS results in the 15-50 ng/mL range and KIMS data resulted in regression coefficients of 0.689 for the research specimens and 0.546 for DOD specimens. The results suggest that the KIMS cannabinoid screening assay is deficient in detecting positives around the cutoff (15-25 ng/mL THC-COOH). This limitation of the KIMS cannabinoid screening method compromises the identification of true positive specimens, therefore reducing the effectiveness of the assay. The success of the DOD program is dependent on sensitive and specific screening assays; the high prevalence of false-negative cannabinoid results compromises the program's primary objective of drug deterrence.

Extraction of benzoylecgonine from urine specimens with Cerex Polycrom Clin II solid-phase extraction columns and the Speedisk Pressure Processor.

Benzoylecgonine (BZE) extraction from urine was explored using Cerex Polycrom Clin II solid-phase extraction (SPE) columns and the Speedisk 48 Pressure Processor as an alternative to the Prep1 automated sample processor and XTRX Type RP/W columns. Linearity for urine standards extracted using the Cerex-Speedisk method ranged from 20 to 3000 ng/mL. The mean recovery at the 100-ng/mL cutoff for three lots of columns was 92%. The mean of the within-run means for three batches, which had coefficients of variations of 1.8% or less, was 101.3 ng/mL at the 100-ng/mL cutoff level. Forty-six specimens known to contain BZE were analyzed by both the Prep1-Type RP/W and Cerex-Speedisk methods. The correlation for specimen BZE concentrations between the two methods gave an r2 of 0.9999 and a slope of 1.03. The Cerex-Speedisk system is an inexpensive alternative to the Prep1-Type RP/W system. It is less costly, requires little maintenance, has a small footprint, is hood compatible, and can process four times the number of specimens in a given time.

Plasmid and chromosomal basis of tolerance to cadmium and resistance to antibiotics in normal bovine duodenal bacterial flora.

Cadmium (Cd) tolerance and antibiotic resistance was studied in duodenal flora of 20 normal bovine samples. Twelve bacterial isolates (5 Staphylococcus spp, 4 Enterococcus faecalis, 2 Bacillus spp, and a Pseudomonas sp) were grown in Luria broth containing 0.05 to 0.8 mM of cadmium chloride (CdCl). All isolates displayed multiple antibiotic resistance, with 2 Enterococcus strains and Pseudomonas pickettii demonstrating resistance to 12/17 antibiotics tested. With the exception of Staphylococcus sp, all contained plasmid DNA. Curing to remove plasmid DNA determined if Cd tolerance and/or antibiotic resistance was plasmid or chromosomally mediated. None of the bacteria became sensitive to CdCl after curing, suggesting that tolerance was not plasmid-mediated. Six bacteria became sensitive to antibiotics after curing indicating that antibiotic2 resistance was plasmid mediated. Two of these bacteria became sensitive to multiple antibiotics; a Staphylococcus sp became sensitive to ampicillin, ceftiofur and cephalothin, and a Enterococcus strain became sensitive to neomycin, oxacillin, and tiamulin. All of the isolates were probed for the presence of known Cd-resistance genes (cadA, cadC, and cadD). DNA-DNA hybridization revealed cadA- and cadC-related sequences in chromosomal DNA of a Staphylococcus sp, an Enterococcus strain, and in plasmid DNA of another Staphylococcus sp. No cadD-related sequences were detected in any of the 12 isolates even under reduced stringency of hybridization.

Pseudomonas pickettii: a common soil and groundwater aerobic bacteria with pathogenic and biodegradation properties.

Pseudomonas pickettii is an aerobic, nonfermentative, Gram-negative rod-shaped, bacterium that has been isolated from soil, water, humans, and recently the bovine intestinal tract. It belongs to the rRNA group II of the genus Pseudomonas and has three biovars: Va-1, Va-2, and biovar 3/thomasii. P. pickettii can cause pneumonia, meningitis, endocarditis, and osteomyelitis in humans. It frequently is associated with nosocomial infections that often are linked to contaminated injectable solutions. P. pickettii exhibits remarkable ability to degrade a variety of toxic compounds such as chlorophenols, aromatic hydrocarbons, 2,4-dichlorophenoxyacetic acid, and pentacyclic triterpeniod compounds. The genes that encode for these properties are chromosome- and plasmid-associated. Strains of the organism also have demonstrated resistance to heavy metals, such as cadmium, copper, and zinc. This species can survive in a nutrient-poor environment and use a variety of toxic compounds as carbon and energy sources, making it an ideal candidate for study in the biodegradation of toxic compounds found in wastewater and soils.

Microbial resistance to metals in the environment.

Many microorganisms demonstrate resistance to metals in water, soil and industrial waste. Genes located on chromosomes, plasmids, or transposons encode specific resistance to a variety of metal ions. Some metals, such as cobalt, copper, nickel, serve as micronutrients and are used for redox processes, to stabilize molecules through electrostatic interactions, as components of various enzymes, and for regulation of osmotic pressure. Most metals are nonessential, have no nutrient value, and are potentially toxic to microorganisms. These toxic metals interact with essential cellular components through covalent and ionic bonding. At high levels, both essential and nonessential metals can damage cell membranes, alter enzyme specificity, disrupt cellular functions, and damage the structure of DNA. Microorganisms have adapted to the presence of both nutrient and nonessential metals by developing a variety of resistance mechanisms. Six metal resistance mechanisms exist: exclusion by permeability barrier, intra- and extra-cellular sequestration, active transport efflux pumps, enzymatic detoxification, and reduction in the sensitivity of cellular targets to metal ions. The understanding of how microorganisms resist metals can provide insight into strategies for their detoxification or removal from the environment.