The emergence of multi-drug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) causes a major threat to public health due to its limited therapeutic options. There is an urgent need for the development of new effective antimicrobial agents and alternative strategies that are effective against resistant bacteria. The parallel legalization of cannabis and its products has fueled research into its many therapeutic avenues in many countries around the world. This study aimed at the development of a reliable method for the extraction, purification, characterization, and quantification of cannabidiolic acid (CBDA) and its decarboxylated form cannabidiol (CBD) present in the fiber type Cannabis sativa L. The two compounds were extracted by ethanol, purified on a C18 sep-pack column, and the extracts were analyzed by high performance liquid chromatography coupled with ultraviolet (UV)–vis and ESI-MS (electrospray ionization mass spectrometry) detection. The antimicrobial effect of CBDA and CBD was also evaluated. CBD displayed a substantial inhibitory effect on Gram-positive bacteria with minimal inhibitory concentrations ranging from 1 to 2 µg/mL. Time kill analysis and minimal bactericidal concentration revealed potential bactericidal activity of CBDA and CBD. While cannabinoids showed a significant antimicrobial effect on the Gram-positive S. aureus and Staphylococcus epidermidis, no activity was noticed on Gram-negative Escherichia coli and Pseudomonas aeruginosa. CBDA presented a two-fold lower antimicrobial activity than its decarboxylated form, suggesting that the antimicrobial pharmacophore of the analyzed cannabinoids falls in the ability for permeabilizing the bacterial cell membrane and acting as a detergent-like agent. A synergy test performed on MRSA with CBD and a range of antibiotics did not indicate a synergetic effect, but noteworthy no antagonist influence either. CBD and CBDA manifested low hemolytic activity on human red blood cells. Likewise, the safety of CBD toward human keratinocyte cells presents no toxicity at a concentration of up to seven-fold higher than the antibacterial minimal inhibitory concentration. Similarly, both CBD and CBDA are well tolerated by mammals, including humans, and conserve a safe value limits for blood-contacting drug development. Overall, CBD exhibited a strong antimicrobial effect against Gram-positive strains and could serve as an alternative drug for tackling MRSA.

Enteroaggregative Escherichia coli (EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with traveler's diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termed agg5A. The sequence of the agg5DCBA gene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of the agg3A gene. The complete agg5DCBA gene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherent E. coli HB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found the agg5A gene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant. During the past decades, enteroaggregative Escherichia coli (EAEC) has emerged as an important pathogen, causing diarrhea in adults and children in both industrialized and nonindustrialized countries (1-5). Moreover, EAEC has also been linked to diarrheal outbreaks (6, 7) including a recent outbreak of foodborne hemorrhagic colitis in Germany affecting more than 4,000 individuals and resulting in a high case fatality rate (8, 9). Additionally, an EAEC urinary tract infection-related outbreak was reported in Denmark (10). Nevertheless, despite EAEC implications in several clinical scenarios, the molecular epidemiology of this pathogen remains unclear. This is mostly due to the heterogeneity of strains, and even though several virulence genes have been identified in EAEC, none have shown to be present in all strains (11)(12)(13)(14), making the recognition of truly virulent strains difficult.Several reports suggest that the key step in EAEC pathogenesis is the ability of the pathogen to adhere to and colonize the intestinal tract, which in EAEC prototype strains is facilitated by aggregative adherence fimbriae (AAF), followed by heavy biofilm formation (15-18). Four variants of the AAF major structural subunit have been described so far: AggA (AAF/I), AafA (AAF/II), Agg3A (AAF/III), and Agg4A (AAF/IV), all regulated by the transcriptional activator AggR, situated on the EAEC virulence plasmid pAA (19)(20)(21)(22)(23). AAFs are distantly related to the Dr family of adhesins, whose biogenesis requires a dedicated periplasmic chaperone, an out...

No abstract