Investigation of microorganisms in cannabis after heating in a commercial vaporizer.

Introduction: There are concerns about microorganisms present on cannabis materials used in clinical settings by individuals whose health status is already compromised and are likely more susceptible to opportunistic infections from microbial populations present on the materials. Most concerning is administration by inhalation where cannabis plant material is heated in a vaporizer, aerosolized, and inhaled to receive the bioactive ingredients. Heating to high temperatures is known to kill microorganisms including bacteria and fungi; however, microbial death is dependent upon exposure time and temperature. It is unknown whether the heating of cannabis at temperatures and times designated by a commercial vaporizer utilized in clinical settings will significantly decrease the microbial loads in cannabis plant material. Methods: To assess this question, bulk cannabis plant material supplied by National Institute on Drug Abuse (NIDA) was used to assess the impact of heating by a commercial vaporizer. Initial method development studies using a cannabis placebo spiked with Escherichia coli were performed to optimize culture and recovery parameters. Subsequent studies were carried out using the cannabis placebo, low delta-9 tetrahydrocannabinol (THC) potency and high THC potency cannabis materials exposed to either no heat or heating for 30 or 70 seconds at 190°C. Phosphate-buffered saline was added to the samples and the samples agitated to suspend the microorganism. Microbial growth after no heat or heating was evaluated by plating on growth media and determining the total aerobic microbial counts and total yeast and mold counts. Results and discussion: Overall, while there were trends of reductions in microbial counts with heating, these reductions were not statistically significant, indicating that heating using standard vaporization parameters of 70 seconds at 190°C may not eliminate the existing microbial bioburden, including any opportunistic pathogens. When cultured organisms were identified by DNA sequence analyses, several fungal and bacterial taxa were detected in the different products that have been associated with opportunistic infections or allergic reactions including Enterobacteriaceae, Staphylococcus, Pseudomonas, and Aspergillus.

Proteolytic activity by multiple bacterial species isolated from chronic venous leg ulcers degrades matrix substrates.

BACKGROUND: A major feature of chronic wounds is the loss of tissue, with the exposure of dermal components preventing primary closure and leading to bacterial colonization. Bacterial colonization has been proposed as one of the common underlying pathologies present in chronic wounds. The objective of this exploratory study was to identify bacteria cultured from chronic venous leg ulcers and test for proteolytic activity that degrades matrix substrates. METHOD: Bacteria were isolated, cultured, and identified from six subjects (average age = 62.8 years) over 2-10 months under an approved protocol using swabs and microbiological culture media. Proteolytic activity against (a) gelatin, (b) an elastin substrate, and (c) a serine/trypsin-sensitive substrate was determined using a colorimetric plate assay with an ELISA plate reader and zymography. RESULTS: We identified 13 bacteria that expressed proteolytic activity against one or more of the tested substrates. Of these, six were Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Corynebacterium, and Streptococcus bovis) and seven were Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Morganella morganii, Klebsiella pneumoniae, Bacteroides fragilis, and Serratia marcescens) organisms. Two of these, S. aureus and P. aeruginosa, are recognized wound pathogens. CONCLUSIONS: Multiple bacteria species isolated from colonized venous leg ulcers have the capacity to secrete proteases capable of degrading components of the extracellular matrix important for wound healing. Matrix degradation by bacteria may contribute to delays in tissue deposition and repair, suggesting that treatment of chronic wounds should include appropriate management of colonizing bacteria.

The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1-->4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1-->6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.